flyDIVaS: A Comparative Genomics Resource for Drosophila Divergence and Selection

Abstract

With arguably the best finished and expertly annotated genome assembly, Drosophila melanogaster is a formidable genetics model to study all aspects of biology. Nearly a decade ago, the 12 Drosophila genomes project expanded D. melanogaster’s breadth as a comparative model through the community-development of an unprecedented genus- and genome-wide comparative resource. However, since its inception, these datasets for evolutionary inference and biological discovery have become increasingly outdated, outmoded, and inaccessible. Here, we provide an updated and upgradable comparative genomics resource of Drosophila divergence and selection, flyDIVaS, based on the latest genomic assemblies, curated FlyBase annotations, and recent OrthoDB orthology calls. flyDIVaS is an online database containing D. melanogaster-centric orthologous gene sets, CDS and protein alignments, divergence statistics (% gaps, d_N, d_S, d_N/d_S), and codon-based tests of positive Darwinian selection. Out of 13,920 protein-coding D. melanogaster genes, ∼80% have one aligned ortholog in the closely related species, D. simulans, and ∼50% have 1–1 12-way alignments in the original 12 sequenced species that span over 80 million yr of divergence. Genes and their orthologs can be chosen from four different taxonomic datasets differing in phylogenetic depth and coverage density, and visualized via interactive alignments and phylogenetic trees. Users can also batch download entire comparative datasets. A functional survey finds conserved mitotic and neural genes, highly diverged immune and reproduction-related genes, more conspicuous signals of divergence across tissue-specific genes, and an enrichment of positive selection among highly diverged genes. flyDIVaS will be regularly updated and can be freely accessed at www.flydivas.info. We encourage researchers to regularly use this resource as a tool for biological inference and discovery, and in their classrooms to help train the next generation of biologists to creatively use such genomic big data resources in an integrative manner.

Rates of phenotypic divergence vary greatly between functional classes. In many cases, functional divergence reflects the evolutionary rates of their underlying genes and proteins (Castillo-Davis et al. 2004; Lemos et al. 2005; Janecka et al. 2012). For example, conserved cellular processes such as growth, metabolism, and replication are encoded by some of the slowest evolving genes, alignable across kingdoms (Zhang and Li 2004; Peregrín-Alvarez et al. 2009). At the other end of the divergence spectrum, rapidly evolving immune-related genes in animals underlie highly dynamic host-parasite interactions that often lack traceable orthologs (Clark and Lazzaro 2012). Similarly, fast evolving sex-related genes code for highly diverged traits involved in sexual dimorphism, reproductive isolation, and species differences and are common across sexual taxa (Wyckoff et al. 2002; Swanson and Vacquier 2002; Singh et al. 2012).

While the comparison of aligned sequences between species provides a complementary molecular approach to study organismal diversity, it also differentiates the two faces of selection—negative and positive—acting on biological processes. Patterns of nucleotide divergence tell us much about the fitness effects of mutational perturbations on proteins and their associated functional systems. Extending our current comparative framework to the level of the codon, both the strength and direction of selection can be inferred by comparing the ratio of nonsynonymous substitutions per nonsynonymous site (d_N) to synonymous substitutions per synonymous site (d_S) (Li et al. 1985; Saitou and Nei 1987). Genes harboring low d_N/d_S ratios reflect high levels of protein conservation across species as negative selection preserves protein function for conserved processes. Genes with orthologous codons exhibiting high d_N/d_S suggest that positive selection quickly drives the fixation of amino acids as organisms better adapt to their surroundings and to each other. Alternatively, high d_N/d_S may indicate a less substantive role of selection on preserving protein function.

The increasing availability of genome assemblies has now made this molecular evolutionary framework a cornerstone of comparative and functional genomic analysis. Coupled with ever-expanding functional annotations (e.g., gene ontologies, tissue, developmental stage, etc.), we have increasing power to detect divergence signatures across biological processes. In fact, some of the most interesting findings from genome projects are the validation and/or discovery of new evolutionary patterns that illuminate the adaptive history of the sequenced species (e.g., Stapley et al. 2010; Radwan and Babik 2012). Various substitution models of d_N/d_S evolution can also yield insight into the site heterogeneity of protein stasis and change, thus providing solutions to diverse biological problems ranging from conservation management (Crandall et al. 2000; Stockwell et al. 2003) to drug targeting and production (Allen et al. 2014). However, this comparative functional framework depends on a highly accurate set of assemblies with precise gene models.

Over the last 15 yr, Drosophila has transformed from a premiere genetic model into among the most powerful genomic models with unprecedented resources and tools for comparative (Clark et al. 2007), population (Mackay et al. 2012), and functional (Celniker et al. 2009; Robinson et al. 2013; dos Santos et al. 2015) genomics. D. melanogaster was among the first eukaryotes with a “finished” genome (Hoskins et al. 2015) and expertly curated gene models across the phylogeny (dos Santos et al. 2015). Over a decade ago, fruit fly researchers from diverse fields collaborated to assemble, align, and annotate a dozen species of Drosophila spanning 80 million yr of evolution (Clark et al. 2007). An online resource known as the Drosophila AAA (Assembly, Alignment, Annotation) site was developed and curated by the Drosophila community as a temporary measure to provide immediate community access to this unique comparative genomics resource. As of 2016, over 1200 papers have cited the original Clark et al. (2007) publication, and researchers continue to analyze results from this important dataset even though D. melanogaster has undergone two major genomic assembly revisions, numerous genomic releases in the other sequenced species, and more than 50 annotation updates.

Here, we present an updated, comprehensive comparative genomics resource of divergence and selection on protein-coding genes in 12 species of the genus Drosophila. flyDIVaS will be regularly updated in synchrony with the latest gene models from FlyBase and orthology calls from OrthoDB. Users will be able to choose between four taxonomic datasets (Figure 1) covering different phylogenetic and sequence depths. flyDIVaS_v1 provides: 1) 1:1 orthologous gene sets, 2) CDS and protein alignments including gap-masked alignments, 3) d_N/d_S estimates, and 4) results from codon substitution (site-specific) selection tests. Alignments and their resulting phylogenetic trees can be visualized online through interactive graphical features. We also present a preliminary analysis of divergence and selection across functional ontological categories and confirm previous observations of high immune and reproductive gene divergence, with stronger signal in genes that are tissue-specific. While highly diverged proteins are enriched in positive selection in the testis and ovary, they appear to be neutrally evolving in accessory glands. Our primary objective is to provide both researchers and students a freely available, gold-standard platform to explore the divergence and adaptive landscape across nearly 100 million yr of evolution.

Figure 1.

Phylogeny and taxonomic datasets of the 12 Drosophila species used in flyDIVaS. Species are organized into four major taxonomic group indicated by color: D. melanogaster and D. simulans (n = 2, red), melanogaster subgroup (n = 5, light blue), melanogaster species group (n = 6, gray), 12 Drosophila genome species (n = 12, dark blue). Males (right) and females (left) of each species are presented and scaled according to their relative size (images generated by Nicolas Gompel).

Materials and Methods

Data source

Coding sequences (CDS and their translated protein) of the longest transcript were downloaded from FlyBase R2015_2 (http://www.flybase.org) for each of the 12 sequenced Drosophila species (Clark et al. 2007): D. melanogaster, D. simulans, D. sechellia, D. yakuba, D. erecta, D. ananassae, D. pseudoobscura, D. persimilis, D. willistoni, D. mojavensis, D. virilis, and D. grimshawi. These data include the latest genomic release of D. simulans (Hu et al. 2013) and updated FB2015_02 annotations from NCBI’s GNOMON annotation pipeline (Souvorov et al. 2010), which integrates new RNAseq data for nine of the 12 species (Dmel, Dsim, Dyak, Dere, Dana, Dpse, Dwil, Dmoj, and Dvir). The number of unique CDS per species range from 13,920 in D. melanogaster to 16,466 in D. sechellia.

Species groupings

Due to divergence and incomplete genome assemblies, greater phylogenetic depth generally results in less alignment coverage. To provide users with a selection of species depths and sequence coverages, we generated four taxonomic datasets (Figure 1): 1) Dmel-Dsim; 2) mel subgroup: Dmel, Dsim, Dsec, Dyak, and Dere; 3) mel group: Dmel, Dsim, Dsec, Dyak, Dere, and Dana; and 4) twelve species: Dmel, Dsim, Dsec, Dyak, Dere, Dana, Dpse, Dper, Dwil, Dmoj, Dvir, and Dgri. The Dmel-Dsim species group offers the greatest genomic coverage of 11,278 1:1 orthologs spanning ∼18.6 Mbp, ∼15% of the entire D. melanogaster euchromatic genome, while the 12 species set contains 6,040 1:1 orthologs covering ∼11.76 Mbp (Table 1).

Table 1. Summary of the four taxonomic datasets used in flyDIVaS_v1.1.

Taxonomic Dataset	Number of 1:1 Orthologs	Mean Alignment Coverage (%)	Mean dN/dS (ω)	Positively Selected Gene Fraction (%)
				M1a vs. M2a (FDR adjusted)	M7 vs. M8 (FDR adjusted)	M8 vs. M8a (FDR adjusted)
D. melanogaster and D. simulans	11,278	98.6	0.205	NA	NA	NA
melanogaster subgroup	9,169	95.0	0.129	3.3 (2.4)	4.2 (3.0)	0.2 (0.1)
melanogaster group	8,649	92.1	0.086	2.3 (1.6)	4.4 (3.7)	3.7 (2.2)
Drosophila 12 species	6,040	83.9	0.065	0.8 (0.4)	9.6 (5.3)	2.3 (1.0)

Included are orthology and alignment coverages, divergence estimates (d_N/d_S), and fractions of positive selected genes fitted to nested PAML (Yang 1997) models of selection.

Alignment and analysis

OrthoDB-derived D. melanogaster based orthologies for the 12 species were downloaded from FlyBase (Waterhouse et al. 2013; dos Santos et al. 2015) (gene_orthologs_fb_2015_02.tsv). For each of the four taxonomic groupings, 1:1 D. melanogaster pairwise orthologs were collected for divergence and selection analyses. Only genes with a single 1:1 ortholog for each species in that particular dataset were used. For example, the well known developmental gene, decapentaplegic (dpp), has a single ortholog in all 12 Drosophila species, and subsequently has both alignments and analyses for each of the four taxonomic groupings in flyDIVaS. On the other hand, the commonly studied gene, Alcohol dehydrogenase (Adh), has a duplication in D. yakuba and, thus, is not present in any taxonomic datasets other than the Dmel-Dsim grouping. A summary of the four 1:1 orthology datasets is found in Table 1.

For each taxonomic dataset, CDS was translated, and sequences were aligned using default parameters in MUSCLE v3.8.31 (Edgar 2004). Amino acid alignments were back-translated to the original CDS sequences and gap-adjusted via perl scripts to retain inframe codons. To reduce alignment errors surrounding insertions and deletions that can negatively affect protein divergence and selection analyses (Markova-Raina and Petrov 2011), we masked +/− three flanking nucleotides at each indel with Ns. Alignment statistics are found in Table 1, and the three generated alignment sets (protein unmasked, CDS unmasked, and CDS masked), as well as unaligned raw fasta files, are available via batch download at flyDIVaS (see below).

Estimates of protein divergence and phylogenetic tests of selection are based on a codon substitution framework implemented by PAML (Yang 1997). Rates of CDS/protein evolution (d_N; d_S; d_N/d_S, often referred to as ω) were estimated using PAML model, M0. Tests for selection on protein-coding regions compared three nested pairs of site-specific models: 1) model M1a (neutral) vs. M2a (positive selection), 2) model M7 (beta-distributed) vs. M8 (beta+ω>1) (Yang 1997), and 3) model M8 (beta+ω>1) vs. model M8a (beta+ω=1) (Swanson et al. 2003; Wong et al. 2004). Confidence values for model comparisons were generated using a likelihood ratio test (LRT) against a χ² distribution. False Discovery Rates (FDR) were generated using the q-value package in R (Storey et al. 2015), with significance determined via a corrected P-value < 0.01. Figure 2 provides a schematic of the flyDIVaS workflow. We stress that divergence estimates and selection tests using the 12 Drosophila species dataset should be met with caution due to the saturation of d_S at this phylogenetic distance (see Box 2 in Larracuente et al. 2008).

Figure 2.

Data flow and analyses in flyDIVaS. This database of Drosophila divergence and selection is based on 1:1 orthology calls of curated CDS fasta files from species of the 12 Drosophila genome project (Clark et al. 2007) referenced against D. melanogaster.

Database architecture

flyDIVaS_v1 was developed using an open-source bootstrap architecture, and promotes an interactive user experience through multiple JavaScript plugins. The database is easily updateable and extensible due to an object- (i.e., gene-) centric data structure. The gene-centric schema also decreases computational time required client-side since data files are neither large nor complex. We use a newly available library of open-source JavaScript plugins called BioJS (https://www.biojs.net). These bioinformatics plugins include client-based tools that allow the user to quickly scan the alignment and visualize the percentage conservation at each site. Additionally, we provide an interactive BioJS neighbor-joining tree plugin with collapsible internal nodes. For users with basic informatics skills, flyDIVaS provides complete alignment sets (both pre and postmasked alignment files are provided, as are unaligned raw fasta files) and divergence and PAML analysis results for each taxonomic dataset on the Downloads page.

Data availability

All data necessary for confirming the conclusions presented in the article are represented fully within the article and at the flyDIVaS website. The flyDIVaS database is freely available for noncommercial use at http://www.flydivas.info.

Results and Discussion

flyDIVaS: Divergence and selection in Drosophila

The genus Drosophila provides an ideal model to study the mode and tempo of evolutionary change. Here, we introduce flyDIVaS, a new online resource of divergence and selection on protein-coding regions across the fruit fly genus (Figure 3). With a dozen well-assembled and expertly annotated species, relatively small euchromatic genomes, and conserved synteny, Drosophila offers a rich trove of data with which to elucidate the molecular and evolutionary mechanisms of conservation and divergence. The initial dataset, generated over a decade ago (Clark et al. 2007), was applied to fields as diverse as development, physiology, and cell biology to better understand both pattern and process and, ever since, these data have served as a gold standard for both geneticists and genomicists interested in everything from evolutionary inference to structure–function relationships. Newly assembled species (Hu et al. 2013), more comprehensive RNAseq-based annotations (Chen et al. 2014), and client-based database platforms, offer a unique opportunity to develop a newly updated comparative genomics resource immediately accessible to a wider cast of researchers and research communities.

Figure 3.

flyDIVaS homepage. Search tool allows users to select a Drosophila gene and one of four taxonomic datasets potentially available. Once these parameters are chosen, a summary of the gene and its associated alignment, divergence, orthology, and selection test results are automatically generated. Phylogenetic view changes when a taxonomic dataset is chosen. A summary of orthology, alignment, and divergence is also provided for the chosen dataset. An interactive JavaScript plugin is provided for users to explore alignment characteristics of their selected gene. Features not shown in figure include a gene-specific neighbor-joining tree (Saitou and Nei 1987) of the aligned sequences, and downloadable fasta files. flyDIVaS can be accessed at www.flydivas.info.

flyDIVaS is freely available as a user-friendly online interface (www.flydivas.info). As a comparative genomics resource for discovery, flyDIVaS generates and provides alignments and selection analyses derived from community-curated resources via user-friendly web tools. The home page is designed to quickly return precomputed data for currently annotated D. melanogaster genes using one of four taxonomic datasets of varying phylogenetic depths (2, 5, 6, and 12 species; Figure 3). The user queries a D. melanogaster gene, using an auto-fill search tool, based on current FlyBase synonyms from any of three accession types: FlyBase gene symbol, FBgn, or a “CG number”. The “species” dropdown menu automatically populates according to the extent of a gene’s orthology among the 12 Drosophila species. Once a gene and its associated dataset are chosen, divergence statistics and links are automatically displayed in the “Gene Summary” section. In addition, basic summary statistics for the entire dataset are shown in the “Dataset Summary” section, found directly below the color-coded, layered phylogeny (Figure 1).

Our original intention was to provide a regularly updated portal for researchers to download comprehensive datasets from this unique comparative genomics resource, with users running analyses via their own inhouse tools. However, most geneticists are interested in a finite set of genes, and/or lack the necessary bioinformatics skills to handle large datasets (Pevzner and Shamir 2009; Welch et al. 2014) that are not readily accessible through graphical user interfaces (GUIs). To serve this large segment of the research community, we use the latest offerings of JavaScript tools that are becoming increasingly available to biologists for data integration and visualization. These open-source libraries allow biologists like us, without prior training in web development, to create online portals with the capacity to interactively visualize complex biological data. flyDIVaS uses BioJS, an open-source set of JavaScript libraries, to help visualize biological data across alignments and phylogenetic trees (www.biojs.net). flyDIVaS applies BioJS in two visual components: 1) an alignment viewer, allowing the user to visualize color-coded alignments of the selected gene, and 2) a basic neighbor-joining phylogeny of the selected gene (Saitou and Nei 1987) allowing users to examine individual characteristics of the gene tree including branch lengths and to compare this gene tree with the canonical species tree (Figure 3). Furthermore, for each gene, we provide raw multi-fasta files for download so that users can perform alignments and analyses using their favorite bioinformatic toolkits.

Integrating such web-friendly tools with large complex datasets may also expedite a much-needed pedagogical shift in the way that big data science such as genomics is taught in the classroom. flyDIVaS’ use of client-side processing elicits fast response times and little overhead on the web server, permitting scalable increases in database usage. Users with low broadband width will not suffer from long download times as each precomputed gene file is only ∼4 kB. flyDIVaS is particularly compatible with mobile and tablet devices providing accessible platforms in which students and scientists can readily explore comparative and evolutionary analysis results “on the fly”.

In addition to gene-specific queries, flyDIVaS provides bulk download access for informatics-savvy users to examine these data, en masse. A tarball (tar.gz) for each of the four taxonomic datasets is available on the “Downloads” page. Included are compressed sets of multi-fasta files for each alignment (both masked and unmasked) as well as raw CDS fasta files. flyDIVaS also provides tab-separated tables consisting of analysis results for the selection-based models including likelihood values for each of the models, chi-square statistics from the likelihood ratio tests, and both regular and adjusted P-values for the model comparisons. The documentation file, “README_flyDIVaS.txt”, found on the Downloads page in flyDIVaS, details the analysis parameters provided.

A major challenge in maintaining an up-to-date and topical genomic database is handling the constant moving targets of updated genome assemblies and annotations. flyDIVaS uses an automated pipeline to directly download standardized data from FlyBase for both orthology relationships (originally from OrthoDB) and annotated CDS sequences from the original 12 species. We plan to provide a major release each year, in consultation with FlyBase, with potential new offerings such as evolutionary rate covariation (e.g., Clark et al. 2012), network connectivity statistics, and lineage-specific tests of selection, depending on users’ needs.

Conservation and divergence in Drosophila

Evolutionary rates vary greatly among genes and the proteins they encode. flyDIVaS, based on the best assembled and annotated genomes, serves as a foundational data resource for biological discovery. In this next section, we provide a precursory and annotated functional survey of genus-wide divergence and adaptive landscape using flyDIVaS data. In each of the four taxonomic datasets, protein divergence is unimodally distributed, but heavily skewed with proteins dispersed along a relatively long tail of high divergence. As the number of species and overall phylogenetic depth increases, both mean d_N and d_S increase (Supplemental Material, Figure S1 and Figure S2) while mean d_N/d_S remains relatively constant (Figure S3), as expected. However, it is clear that the inclusion of more species reduces the overall variance in divergence estimates (Figure S4), highlighting the power of dense phylogenetic coverage such as the data provided by the 12 species dataset (e.g., distribution of P-values for tests of positive selection: Figure S5, Figure S6, Figure S7, and Figure S8).

Our extensive survey of ontologies and tissues also demonstrate that mean rates of amino acid change vary across functional classes (Figure 4A). A large variety of gene ontological (GO) categories are conserved in biological processes (Figure S9), molecular function (Figure S10), and cellular components (Figure S11), as well as FlyBase-defined organismal and developmental ontologies (Figure S12 and Figure S13). Neural tissues including the brain, thoracicoabdominal ganglia, head, and eye contain more conserved genes on average (Figure 4B). In the six species dataset, the most conserved genes with a d_N/d_S of zero (i.e., no replacement changes) are enriched for mitotic and cell cycle processes, as seen in other taxa (Castillo-Davis et al. 2004).

Figure 4.

Functional analysis of divergence. (A) Gene ontology comparison for each of the four datasets showing the individual distributions of d_N/d_S in the top hierarchical ontological categories of Biological Process and rank ordered according to divergence. (B) Ranked distribution of d_N/d_S in selected tissues in the melanogaster group dataset. Genes labeled as “general” are expressed in > 50% of the examined tissues (open) and genes labeled as “specific” are expressed in a single tissue (gray). Tissue expression data taken from FlyAtlas (Robinson et al. 2013). Astericks denote significant difference between general and specific tissue-expressed genes; ** P < 0.001, Wilcoxon rank-sum test.

Mean evolutionary rate variation among functional ontologies and tissues appears to be driven by the disproportional presence of highly diverged genes in each functional class (Figure 4, Figure S9, Figure S10, Figure S11, Figure S12, Figure S13, Figure S14, Figure S15, Figure S16, and Figure S17). Such rapidly evolving genes and their associated functional classes may play an important role in species-specific differences due to a greater relaxation of selection or adaptation (Singh et al. 2012). As reported in previous literature, immune and reproductive ontological classes are among the most rapidly evolving functional groups (Figure 4A and Figure S9). Immune-related genes are hypothesized to coevolve through a continuous arms race with parasitic invaders (Singh and Kulathinal 2000; Wyckoff et al. 2002; Schlenke and Begun 2003; Sackton et al. 2007; Singh et al. 2012). Extracellular proteins—a large component of both immune and reproductive systems—are the most rapidly evolving cellular component class (Figure S13). In addition, the most diverged tissues include such male reproductive tissue as accessory glands and testes (Figure 4, Figure S14, Figure S15, Figure S16, and Figure S17), both involved in sperm development and maturation, and a major component in sperm competition (Clark et al. 1995). In fact, the top 10% rapidly evolving proteins are enriched for genes that are upregulated in the testis (P < 0.001; χ² ≈ 114.5).

While our results confirm a landscape of functional divergence that highlights the rapid evolution of immune- and reproductive-related traits (Haerty et al. 2007; Sackton et al. 2007; Singh et al. 2012), signals of divergence are strengthened when comparing tissue-specific genes. Figure 4B confirms previous studies in both mammals and Drosophila revealing a larger range of mean d_N/d_S estimates among tissue-specific genes compared to genes coexpressed in other tissues (Duret and Mouchiroud 2000; Zhang and Li 2004; Haerty et al. 2007; Meisel 2011). For example, the subset of genes solely expressed in a single reproductive tissue (e.g., accessory gland-specific, testis-specific, ovary-specific) has a significantly larger mean d_N and d_N/d_S than genes that are expressed in the same tissue and coexpressed in other tissues (Figure 4B, Figure S14, Figure S15, Figure S16, and Figure S17). On the other end of the distribution, brain-specific genes are less diverged, in agreement with studies in mammals (Duret and Mouchiroud 2000; Wang et al. 2007).

The higher tissue-specific divergence pattern can be explained by two alternative hypotheses: 1) less functional pleiotropic constraints, or 2) stronger positive selection. Supporting the latter hypothesis, we found a significant enrichment of positively selected genes in the highest 10% of diverged genes in terms of d_N (M7 vs. M8: P < 0.001; χ² ≈ 12.7, M7 vs. M8a: P < 0.001; χ² ≈ 35.0) and d_N/d_S (M7 vs. M8: P < 0.001; χ² ≈ 48.2, M7 vs. M8a: P < 0.001; χ² ≈ 96.2) based on the same site-specific phylogenetic selection models (M7vM8 and M7vM8a), and using the six-species melanogaster group dataset. However, whether this adaptive enrichment is driven by biased detection power due to a greater number of substitutions remains to be tested. An enrichment analysis also found a significant overrepresentation of positive selection in testis-specific genes in the M7 vs. M8 model test (P < 0.01; χ² ≈ 5.5) but not in M7 vs. M8a (P ≈ 0.25; χ² ≈ 1.32). Interestingly, the most rapidly evolving tissue, accessory glands, was not enriched in genes (either general or tissue-specific) evolving under positive selection (M7 vs. M8: P ≈ 0.370; χ² ≈ 0.83, M7 vs. M8a: P ≈ 0.54; χ² ≈ 0.37), indicating a greater role of relaxed selection across this highly divergent class of proteins. Thus, rapidly evolving genes involved in such species-specific traits such as male fertility may be the result of an interplay between neutral and selective forces across a dynamic network of coadapted and newly coopted proteins (Kulathinal and Singh 2012).

Conclusions

D. melanogaster has metamorphosed from a powerful genetic tool into an invaluable genomic model, providing substantive insight across broad biological fields. Much of this transformation was made possible by sequencing related species of the Drosophila phylogeny (Clark et al. 2007), thereby generating a powerful comparative resource to identify novel functional units in D. melanogaster and precipitate new discoveries in evolutionary biology. flyDIVaS provides an updated and updatable database of comparative genomics based on the latest assemblies, orthology calls, and expert, community-based annotations of a dozen phylogenetically diverse fruit flies. At www.flyDIVaS.info, users can access gene-specific divergence and selection profiles or download entire comparative genomics datasets from a choice of four taxonomic groups. A preliminary functional survey supports results from previous literature including highly conserved mitotic, cell cycle and neural genes, the rapid evolution of immune and reproductive genes and genetic systems, strong tissue-specific signatures of divergence, and a role for positive selection in driving amino acid divergence in certain tissues. We strongly encourage users to explore their genes, genetic systems, and fly genomes of interest, and to provide comments and requests to improve flyDIVaS for its next release.