Skeleton phylogeny reconstructed with transcriptomes for the tribe Drosophilini (Diptera: Drosophilidae).

The family Drosophilidae is one of the most important model systems in evolutionary biology. Thanks to advances in high-throughput sequencing technology, a number of molecular phylogenetic analyses have been undertaken by using large data sets of many genes and many species sampled across this family. Especially, recent analyses using genome sequences have depicted the family-wide skeleton phylogeny with high confidence. However, the taxon sampling is still insufficient for minor lineages and non-Drosophila genera. In this study, we carried out phylogenetic analyses using a large number of transcriptome-based nucleotide sequences, focusing on the largest, core tribe Drosophilini in the Drosophilidae. In our analyses, some noise factors against phylogenetic reconstruction were taken into account by removing putative paralogy from the datasets and examining the effects of missing data, i.e. gene occupancy and site coverage, and incomplete lineage sorting. The inferred phylogeny has newly resolved the following phylogenetic positions/relationships at the genomic scale: (i) the monophyly of the subgenus Siphlodora including Zaprionus flavofasciatus to be transferred therein; (ii) the paraphyly of the robusta and melanica species groups within a clade comprised of the robusta, melanica and quadrisetata groups and Z. flavofasciatus; (iii) Drosophila curviceps (representing the curviceps group), D. annulipes (the quadrilineata subgroup of the immigrans group) and D. maculinotata clustered into a clade sister to the Idiomyia + Scaptomyza clade, forming together the expanded Hawaiian drosophilid lineage; (iv) Dichaetophora tenuicauda (representing the lineage comprised of the Zygothrica genus group and Dichaetophora) placed as the sister to the clade of the expanded Hawaiian drosophilid lineage and Siphlodora; and (v) relationships of the subgenus Drosophila and the genus Zaprionus as follows: (Zaprionus, (the quadrilineata subgroup, ((D. sternopleuralis, the immigrans group proper), (the quinaria radiation, the tripunctata radiation)))). These results are to be incorporated into the so-far published phylogenomic tree as a backbone (constraint) tree for grafting much more species based on sequences of a limited number of genes. Such a comprehensive, highly confident phylogenetic tree with extensive and dense taxon sampling will provide an essential framework for comparative studies of the Drosophilidae.

Phylogenetic classification and palm-inflorescence anthophily of the Colocasiomyia zeylanica species group (Diptera: Drosophilidae), with descriptions of five new species.

The zeylanica group is one of the six species groups of the anthophilic genus Colocasiomyia de Meijere in the family Drosophilidae. In addition to two known species, five morphospecies have been recognized as members of this species group but left undescribed formally. In this study, species delimitation of these putatively new species was determined by barcoding of the mitochondrial COI (cytochrome c oxydase subunit I) gene and morphological comparison. Phylogenetic relationships within the genus Colocasiomyia were inferred by a cladistic analysis of 89 morphological characters. Based on the results of these analyses, we redefined the zeylanica species group and established two subgroups within it: the zeylanica subgroup comprised of C. zeylanica, C. nepalensis, C. pinangae sp. nov., C. besaris sp. nov. and C. luciphila sp. nov., and the oligochaeta subgroup of C. oligochaeta sp. nov. and C. grimaldii sp. nov. In addition, we briefly address the anthophilic habits of drosophilid flies using palm (Arecaceae) inflorescences, especially of the zeylanica group, compiling scattered collection records from the Oriental and Papuan regions.

Comparative Mitochondrial Genomes between the Genera Amiota and Phortica (Diptera: Drosophilidae) with Evolutionary Insights into D-Loop Sequence Variability.

To address the limited number of mitochondrial genomes (mitogenomes) in the subfamily Steganinae (Diptera: Drosophilidae), we assembled 12 complete mitogenomes for six representative species in the genus Amiota and six representative species in the genus Phortica. We performed a series of comparative and phylogenetic analyses for these 12 Steganinae mitogenomes, paying special attention to the commonalities and differences in the D-loop sequences. Primarily determined by the lengths of the D-loop regions, the sizes of the Amiota and Phortica mitogenomes ranged from 16,143-16,803 bp and 15,933-16,290 bp, respectively. Our results indicated that the sizes of genes and intergenic nucleotides (IGNs), codon usage and amino acid usage, compositional skewness levels, evolutionary rates of protein-coding genes (PCGs), and D-loop sequence variability all showed unambiguous genus-specific characteristics and provided novel insights into the evolutionary implications between and within Amiota and Phortica. Most of the consensus motifs were found downstream of the D-loop regions, and some of them showed distinct genus-specific patterns. In addition, the D-loop sequences were phylogenetically informative as the data sets of PCGs and/or rRNAs, especially within the genus Phortica.

Revision of the subgenus Stegana (Steganina) from China, with assessment of species delimitation using DNA barcodes (Diptera, Drosophilidae).

A total of 58 (eight known and 50 new) species of the subgenus Stegana (Steganina) from China were surveyed and (re)described: S. (S.) bacilla Chen & Aotsuka, 2004, S. (S.) belokobylskiji Sidorenko, 1997, S. (S.) hirticeps Wang, Gao, & Chen, 2013, S. (S.) izu Sidorenko, 1997, S. (S.) kanmiyai Okada & Sidorenko, 1992, S. (S.) masanoritodai Okada & Sidorenko, 1992, S. (S.) maymyo Sidorenko, 1997, stat. rev., S. (S.) nigripes Zhang & Chen, 2015, S. (S.) alafoliacea Zhang & Chen, sp. nov., S. (S.) baoxing Li & Chen, sp. nov., S. (S.) bibarbata Li & Chen, sp. nov., S. (S.) bimai Cui & Chen, sp. nov., S. (S.) cinereipecta Zhang & Chen, sp. nov., S. (S.) cardua Cui & Chen, sp. nov., S. (S.) cordhirsuta Wang & Chen, sp. nov., S. (S.) cornuta Li & Chen, sp. nov., S. (S.) cucullata Li & Chen, sp. nov., S. (S.) cultella Cui & Chen, sp. nov., S. (S.) curvitabulata Cui & Chen, sp. nov., S. (S.) daiya Cui & Chen, sp. nov., S. (S.) dendrophila Zhang & Chen, sp. nov., S. (S.) flabella Li & Chen, sp. nov., S. (S.) flavipes Li & Chen, sp. nov., S. (S.) formosa Zhang & Chen, sp. nov., S. (S.) fusca Li & Chen, sp. nov., S. (S.) fuscipes Li & Chen, sp. nov., S. (S.) glaucopalpula Cui & Chen, sp. nov., S. (S.) haba Zhang & Chen, sp. nov., S. (S.) hirticlavata Cui & Chen, sp. nov., S. (S.) iaspidea Zhang & Chen, sp. nov., S. (S.) idiasta Cui & Chen, sp. nov., S. (S.) kanda Cui & Chen, sp. nov., S. (S.) labao Li & Chen, sp. nov., S. (S.) lancang Li & Chen, sp. nov., S. (S.) latifoliacea Wang & Chen, sp. nov., S. (S.) liusanjieae Li & Chen, sp. nov., S. (S.) magniflava Cui & Chen, sp. nov., S. (S.) mailangang Li & Chen, sp. nov., S. (S.) marenubila Cui & Chen, sp. nov., S. (S.) menghai Zhang & Chen, sp. nov., S. (S.) menglian Li & Chen, sp. nov., S. (S.) minutiflava Li & Chen, sp. nov., S. (S.) multiprocera Li & Chen, sp. nov., S. (S.) nayun Li & Chen, sp. nov., S. (S.) nigridentata Wang & Chen, sp. nov., S. (S.) nigripalpula Cui & Chen, sp. nov., S. (S.) otphylla Cui & Chen, sp. nov., S. (S.) radiciflava Zhang & Chen, sp. nov., S. (S.) rava Cui & Chen, sp. nov., S. (S.) sciophila Li & Chen, sp. nov., S. (S.) septencolorata Li & Chen, sp. nov., S. (S.) serrata Zhang & Chen, sp. nov., S. (S.) silvestrella Zhang & Chen, sp. nov., S. (S.) simola Cui & Chen, sp. nov., S. (S.) yani Li & Chen, sp. nov., S. (S.) yixiang Zhang & Chen, sp. nov., S. (S.) zaduo Cui & Chen, sp. nov., and S. (S.) zhuoma Cui & Chen, sp. nov. We also provided a complete list of Chinese Steganina species together with their geographical distributions. In addition, the majority of currently available DNA barcode (partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene) sequences of this subgenus (435 sequences of 102 spp.) were employed in a molecular analysis for species delimitation. Taken together, morphology- and molecular-based species delimitation results reached a consensus for an overwhelming majority of these Steganina species (98 of 102 spp.).

Evolution and genomic basis of the plant-penetrating ovipositor: a key morphological trait in herbivorous Drosophilidae.

Herbivorous insects are extraordinarily diverse, yet are found in only one-third of insect orders. This skew may result from barriers to plant colonization, coupled with phylogenetic constraint on plant-colonizing adaptations. The plant-penetrating ovipositor, however, is one trait that surmounts host plant physical defences and may be evolutionarily labile. Ovipositors densely lined with hard bristles have evolved repeatedly in herbivorous lineages, including within the Drosophilidae. However, the evolution and genetic basis of this innovation has not been well studied. Here, we focused on the evolution of this trait in Scaptomyza, a genus sister to Hawaiian Drosophila, that contains a herbivorous clade. Our phylogenetic approach revealed that ovipositor bristle number increased as herbivory evolved in the Scaptomyza lineage. Through a genome-wide association study, we then dissected the genomic architecture of variation in ovipositor bristle number within S. flava. Top-associated variants were enriched for transcriptional repressors, and the strongest associations included genes contributing to peripheral nervous system development. Individual genotyping supported the association at a variant upstream of Gαi, a neural development gene, contributing to a gain of 0.58 bristles/major allele. These results suggest that regulatory variation involving conserved developmental genes contributes to this key morphological trait involved in plant colonization.

Phylogenetic resolution of the fly superfamily Ephydroidea-Molecular systematics of the enigmatic and diverse relatives of Drosophilidae.

The schizophoran superfamily Ephydroidea (Diptera: Cyclorrhapha) includes eight families, ranging from the well-known vinegar flies (Drosophilidae) and shore flies (Ephydridae), to several small, relatively unusual groups, the phylogenetic placement of which has been particularly challenging for systematists. An extraordinary diversity in life histories, feeding habits and morphology are a hallmark of fly biology, and the Ephydroidea are no exception. Extreme specialization can lead to "orphaned" taxa with no clear evidence for their phylogenetic position. To resolve relationships among a diverse sample of Ephydroidea, including the highly modified flies in the families Braulidae and Mormotomyiidae, we conducted phylogenomic sampling. Using exon capture from Anchored Hybrid Enrichment and transcriptomics to obtain 320 orthologous nuclear genes sampled for 32 species of Ephydroidea and 11 outgroups, we evaluate a new phylogenetic hypothesis for representatives of the superfamily. These data strongly support monophyly of Ephydroidea with Ephydridae as an early branching radiation and the placement of Mormotomyiidae as a family-level lineage sister to all remaining families. We confirm placement of Cryptochetidae as sister taxon to a large clade containing both Drosophilidae and Braulidae-the latter a family of honeybee ectoparasites. Our results reaffirm that sampling of both taxa and characters is critical in hyperdiverse clades and that these factors have a major influence on phylogenomic reconstruction of the history of the schizophoran fly radiation.

Evolution and development of male-specific leg brushes in Drosophilidae.

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.

Phortica oldenbergi (Diptera: Drosophilidae): A new potential vector of the zoonotic Thelazia callipaeda eyeworm.

Thelazia callipaeda is a zoonotic nematode parasitizing the eyes of many hosts species, primarily dogs. To date Phortica variegata and Phortica okadai are the only known vectors of this nematode in Europe and China, respectively. In this study we investigated the role played by a third species, Phortica oldenbergi, as vector of T. callipaeda in Europe. Drosophilid flies of this species were collected in central Italy and maintained in laboratory. One hundred forty P. oldenbergi were experimentally infected with T. callipaeda L1 recovered from field collected gravid females belonging to haplotype 1, which is that detected in several countries in Europe. Seventy-four (i.e., 60 females and 14 males) specimens died at 5 days post infection (d.p.i.) (±1) and scored negative for T. callipaeda larvae at the dissection. Sixty-six (i.e., 46 females and 20 males) P. oldenbergi survived and were dissected at 21 d.p.i. From those, T. callipaeda L3 were detected in the proboscis of two females (3.0%). Overall, at the molecular analysis, 11.4% (n = 16/140; 13 females and 3 males) scored positive for the presence of T. callipaeda DNA. Data herein reported brings further insights on the biology of T. callipaeda by adding P. oldenbergi as a new potential intermediate host under experimental conditions. The role of this drosophilid in the transmission cycle of T. callipaeda needs to be confirmed under natural conditions.

Phylogeny of Drosophila saltans group (Diptera: Drosophilidae) based on morphological and molecular evidence.

Drosophila saltans group belongs to the subgenus Sophophora (family Drosophilidae), and it is subdivided into five subgroups, with 23 species. The species in this group are widely distributed in the Americas, primarily in the Neotropics. In the literature, the phylogenetic reconstruction of this group has been performed with various markers, but many inconsistencies remain. Here, we present a phylogenetic reconstruction of the saltans group with a greater number of species, 16 species, which is the most complete to date for the saltans group and includes all subgroups, in a combined analysis with morphological and molecular markers. We incorporated 48 morphological characters of male terminalia, the highest number used to date, and molecular markers based on mitochondrial genes COI and COII. Based on the results, which have recovered the five subgroups as distinct lineages, we propose a new hypothesis regarding the phylogenetic relationships among the subgroups of the saltans group. The relationships of the species within the sturtevanti and elliptica subgroups were well supported. The saltans subgroup showed several polytomies, but the relationship between the sibling species D. austrosaltans and D. saltans and their close relation with D. nigrosaltans were well supported in the molecular and total evidence analyses. The morphological analysis additionally supported the formation of the clade D. nigrosaltans-D. pseudosaltans. The observed polytomies may represent synchronous radiations or have resulted from speciation rates that have been too fast relative to the pace of substitution accumulation.

A new species of the Colocasiomyia gigantea species group (Diptera, Drosophilidae) breeding on inflorescences of Scindapsus maclurei (Araceae).

The gigantea group is one of the six species groups of the genus Colocasiomyia Meijere, 1914 (Diptera, Drosophilidae). All the nine known species of this group breed on inflorescence/infructescence of host plants of the subfamily Monsteroideae (Araceae) and are geographically restricted to the Oriental region: seven species found exclusively from Rhaphidophora spp. in southern China, and the remaining two from Sabah (host plant: Scindapsus coriaceus) in Borneo or from West Java (host plant: Epipremnum pinnatum). In the present paper, a new member of the gigantea group, C. daiae sp. nov., is described, with adult specimens and eggs collected from inflorescences of Scindapsus maclurei in Hainan, China and larvae and pupae reared from field-collected eggs in the laboratory.

Phylotranscriptomics Reveals Discordance in the Phylogeny of Hawaiian Drosophila and Scaptomyza (Diptera: Drosophilidae).

Island radiations present natural laboratories for studying the evolutionary process. The Hawaiian Drosophilidae are one such radiation, with nearly 600 described species and substantial morphological and ecological diversification. These species are largely divided into a few major clades, but the relationship between clades remains uncertain. Here, we present new assembled transcriptomes from 12 species across these clades, and use these transcriptomes to resolve the base of the evolutionary radiation. We recover a new hypothesis for the relationship between clades, and demonstrate its support over previously published hypotheses. We then use the evolutionary radiation to explore dynamics of concordance in phylogenetic support, by analyzing the gene and site concordance factors for every possible topological combination of major groups. We show that high bootstrap values mask low evolutionary concordance, and we demonstrate that the most likely topology is distinct from the topology with the highest support across gene trees and from the topology with highest support across sites. We then combine all previously published genetic data for the group to estimate a time-calibrated tree for over 300 species of drosophilids. Finally, we digitize dozens of published Hawaiian Drosophilidae descriptions, and use this to pinpoint probable evolutionary shifts in reproductive ecology as well as body, wing, and egg size. We show that by examining the entire landscape of tree and trait space, we can gain a more complete understanding of how evolutionary dynamics play out across an island radiation.

Evolution of Olfactory Receptors Tuned to Mustard Oils in Herbivorous Drosophilidae.

The diversity of herbivorous insects is attributed to their propensity to specialize on toxic plants. In an evolutionary twist, toxins betray the identity of their bearers when herbivores coopt them as cues for host-plant finding, but the evolutionary mechanisms underlying this phenomenon are poorly understood. We focused on Scaptomyza flava, an herbivorous drosophilid specialized on isothiocyanate (ITC)-producing (Brassicales) plants, and identified Or67b paralogs that were triplicated as mustard-specific herbivory evolved. Using in vivo heterologous systems for the expression of olfactory receptors, we found that S. flava Or67bs, but not the homologs from microbe-feeding relatives, responded selectively to ITCs, each paralog detecting different ITC subsets. Consistent with this, S. flava was attracted to ITCs, as was Drosophila melanogaster expressing S. flava Or67b3 in the homologous Or67b olfactory circuit. ITCs were likely coopted as olfactory attractants through gene duplication and functional specialization (neofunctionalization and subfunctionalization) in S. flava, a recently derived herbivore.

Male terminalia morphology of sixteen species of the Drosophila saltans group Sturtevant (Diptera, Drosophilidae).

Male terminalia in insects with internal fertilization evolve more rapidly than other structures. The aedeagus is the most variable structure, making it a valuable diagnostic feature to distinguish species. The saltans group Sturtevant of Drosophila Fall contains sibling species, that can be distinguished by their aedeagi. Here, we revised and illustrated the morphology of the male terminalia of the following species: Drosophila prosaltans Duda, 1927; D. saltans Sturtevant, 1916; D. lusaltans Magalhes, 1962; D. austrosaltans Spassky, 1957; D. septentriosaltans Magalhes, 1962; D. nigrosaltans Magalhes, 1962; D. pseudosaltans Magalhes, 1956; D. sturtevanti Duda, 1927; D. lehrmanae Madi-Ravazzi et al., 2021; D. dacunhai Mouro Bicudo, 1967; D. milleri Magalhes, 1962; D. parasaltans Magalhes, 1956; D. emarginata Sturtevant, 1942; D. neoelliptica Pavan Magalhes in Pavan, 1950; D. neosaltans Pavan Magalhes in Pavan, 1950 and D. neocordata Magalhes, 1956. We found that phallic structures (e.g., the aedeagus) evolve more rapidly than periphallic structures (e.g., epandrium), being completely different among the subgroups and within them. This rapid evolution may be due to the action of sexual selection or to the potential role of those structures in speciation.

Comparative mitogenomics of Drosophilidae and the evolution of the Zygothrica genus group (Diptera, Drosophilidae).

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.

Viral susceptibility across host species is largely independent of dietary protein to carbohydrate ratios.

The likelihood of a successful host shift of a parasite to a novel host species can be influenced by environmental factors that can act on both the host and parasite. Changes in nutritional resource availability have been shown to alter pathogen susceptibility and the outcome of infection in a range of systems. Here, we examined how dietary protein to carbohydrate altered susceptibility in a large cross-infection experiment. We infected 27 species of Drosophilidae with an RNA virus on three food types of differing protein to carbohydrate ratios. We then measured how viral load and mortality across species was affected by changes in diet. We found that changes in the protein:carbohydrate in the diet did not alter the outcomes of infection, with strong positive inter-species correlations in both viral load and mortality across diets, suggesting no species-by-diet interaction. Mortality and viral load were strongly positively correlated, and this association was consistent across diets. This suggests changes in diet may give consistent outcomes across host species, and may not be universally important in determining host susceptibility to pathogens.

Competitive history shapes rapid evolution in a seasonal climate.

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.

A phylogenomic study of Steganinae fruit flies (Diptera: Drosophilidae): strong gene tree heterogeneity and evidence for monophyly.

BACKGROUND: The Drosophilidae family is traditionally divided into two subfamilies: Drosophilinae and Steganinae. This division is based on morphological characters, and the two subfamilies have been treated as monophyletic in most of the literature, but some molecular phylogenies have suggested Steganinae to be paraphyletic. To test the paraphyletic-Steganinae hypothesis, here, we used genomic sequences of eight Drosophilidae (three Steganinae and five Drosophilinae) and two Ephydridae (outgroup) species and inferred the phylogeny for the group based on a dataset of 1,028 orthologous genes present in all species (> 1,000,000 bp). This dataset includes three genera that broke the monophyly of the subfamilies in previous works. To investigate possible biases introduced by small sample sizes and automatic gene annotation, we used the same methods to infer species trees from a set of 10 manually annotated genes that are commonly used in phylogenetics. RESULTS: Most of the 1,028 gene trees depicted Steganinae as paraphyletic with distinct topologies, but the most common topology depicted it as monophyletic (43.7% of the gene trees). Despite the high levels of gene tree heterogeneity observed, species tree inference in ASTRAL, in PhyloNet, and with the concatenation approach strongly supported the monophyly of both subfamilies for the 1,028-gene dataset. However, when using the concatenation approach to infer a species tree from the smaller set of 10 genes, we recovered Steganinae as a paraphyletic group. The pattern of gene tree heterogeneity was asymmetrical and thus could not be explained solely by incomplete lineage sorting (ILS). CONCLUSIONS: Steganinae was clearly a monophyletic group in the dataset that we analyzed. In addition to ILS, gene tree discordance was possibly the result of introgression, suggesting complex branching processes during the early evolution of Drosophilidae with short speciation intervals and gene flow. Our study highlights the importance of genomic data in elucidating contentious phylogenetic relationships and suggests that phylogenetic inference for drosophilids based on small molecular datasets should be performed cautiously. Finally, we suggest an approach for the correction and cleaning of BUSCO-derived genomic datasets that will be useful to other researchers planning to use this tool for phylogenomic studies.

Diverse Defenses: A Perspective Comparing Dipteran Piwi-piRNA Pathways.

Animals face the dual threat of virus infections hijacking cellular function and transposons proliferating in germline genomes. For insects, the deeply conserved RNA interference (RNAi) pathways and other chromatin regulators provide an important line of defense against both viruses and transposons. For example, this innate immune system displays adaptiveness to new invasions by generating cognate small RNAs for targeting gene silencing measures against the viral and genomic intruders. However, within the Dipteran clade of insects, Drosophilid fruit flies and Culicids mosquitoes have evolved several unique mechanistic aspects of their RNAi defenses to combat invading transposons and viruses, with the Piwi-piRNA arm of the RNAi pathways showing the greatest degree of novel evolution. Whereas central features of Piwi-piRNA pathways are conserved between Drosophilids and Culicids, multiple lineage-specific innovations have arisen that may reflect distinct genome composition differences and specific ecological and physiological features dividing these two branches of Dipterans. This perspective review focuses on the most recent findings illuminating the Piwi/piRNA pathway distinctions between fruit flies and mosquitoes, and raises open questions that need to be addressed in order to ameliorate human diseases caused by pathogenic viruses that mosquitoes transmit as vectors.

Fly wing evolution explained by a neutral model with mutational pleiotropy.

To what extent the speed of mutational production of phenotypic variation determines the rate of long-term phenotypic evolution is a central question. Houle et al. recently addressed this question by studying the mutational variances, additive genetic variances, and macroevolution of locations of vein intersections on fly wings, reporting very slow phenotypic evolution relative to the rates of mutational input, high phylogenetic signals, and a strong, linear relationship between the mutational variance of a trait and its rate of evolution. Houle et al. found no existing model of phenotypic evolution to be consistent with all these observations, and proposed the improbable scenario of equal influence of mutational pleiotropy on all traits. Here, we demonstrate that the purported linear relationship between mutational variance and evolutionary divergence is artifactual. We further show that the data are explainable by a simple model in which the wing traits are effectively neutral at least within a range of phenotypic values but their evolutionary rates are differentially reduced because mutations affecting these traits are purged owing to their different pleiotropic effects on other traits that are under stabilizing selection. Thus, the evolutionary patterns of fly wing morphologies are explainable under the existing theoretical framework of phenotypic evolution.

Temporal flexibility of gene regulatory network underlies a novel wing pattern in flies.

Organisms have evolved endless morphological, physiological, and behavioral novel traits during the course of evolution. Novel traits were proposed to evolve mainly by orchestration of preexisting genes. Over the past two decades, biologists have shown that cooption of gene regulatory networks (GRNs) indeed underlies numerous evolutionary novelties. However, very little is known about the actual GRN properties that allow such redeployment. Here we have investigated the generation and evolution of the complex wing pattern of the fly Samoaia leonensis We show that the transcription factor Engrailed is recruited independently from the other players of the anterior-posterior specification network to generate a new wing pattern. We argue that partial cooption is made possible because 1) the anterior-posterior specification GRN is flexible over time in the developing wing and 2) this flexibility results from the fact that every single gene of the GRN possesses its own functional time window. We propose that the temporal flexibility of a GRN is a general prerequisite for its possible cooption during the course of evolution.