Resolving between novelty and homology in the rapidly evolving phallus of Drosophila.

The genitalia present some of the most rapidly evolving anatomical structures in the animal kingdom, possessing a variety of parts that can distinguish recently diverged species. In the Drosophila melanogaster group, the phallus is adorned with several processes, pointed outgrowths, that are similar in size and shape between species. However, the complex three-dimensional nature of the phallus can obscure the exact connection points of each process. Previous descriptions based upon adult morphology have primarily assigned phallic processes by their approximate positions in the phallus and have remained largely agnostic regarding their homology relationships. In the absence of clearly identified homology, it can be challenging to model when each structure first evolved. Here, we employ a comparative developmental analysis of these processes in eight members of the melanogaster species group to precisely identify the tissue from which each process forms. Our results indicate that adult phallic processes arise from three pupal primordia in all species. We found that in some cases the same primordia generate homologous structures whereas in other cases, different primordia produce phenotypically similar but remarkably non-homologous structures. This suggests that the same gene regulatory network may have been redeployed to different primordia to induce phenotypically similar traits. Our results highlight how traits diversify and can be redeployed, even at short evolutionary scales.

Large-scale analysis of Drosophila core promoter function using synthetic promoters.

The core promoter plays a central role in setting metazoan gene expression levels, but how exactly it "computes" expression remains poorly understood. To dissect its function, we carried out a comprehensive structure-function analysis in Drosophila. First, we performed a genome-wide bioinformatic analysis, providing an improved picture of the sequence motifs architecture. We then measured synthetic promoters' activities of ~3,000 mutational variants with and without an external stimulus (hormonal activation), at large scale and with high accuracy using robotics and a dual luciferase reporter assay. We observed a strong impact on activity of the different types of mutations, including knockout of individual sequence motifs and motif combinations, variations of motif strength, nucleosome positioning, and flanking sequences. A linear combination of the individual motif features largely accounts for the combinatorial effects on core promoter activity. These findings shed new light on the quantitative assessment of gene expression in metazoans.

The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila.

Sexually dimorphic traits play key roles in animal evolution and behavior. Little is known, however, about the mechanisms governing their development and evolution. One recently evolved dimorphic trait is the male-specific abdominal pigmentation of Drosophila melanogaster, which is repressed in females by the Bric-à-brac (Bab) proteins. To understand the regulation and origin of this trait, we have identified and traced the evolution of the genetic switch controlling dimorphic bab expression. We show that the HOX protein Abdominal-B (ABD-B) and the sex-specific isoforms of Doublesex (DSX) directly regulate a bab cis-regulatory element (CRE). In females, ABD-B and DSX(F) activate bab expression whereas in males DSX(M) directly represses bab, which allows for pigmentation. A new domain of dimorphic bab expression evolved through multiple fine-scale changes within this CRE, whose ancestral role was to regulate other dimorphic features. These findings reveal how new dimorphic characters can emerge from genetic networks regulating pre-existing dimorphic traits.

Enhancer evolutionary co-option through shared chromatin accessibility input.

The diversity of forms in multicellular organisms originates largely from the spatial redeployment of developmental genes [S. B. Carroll, Cell 134, 25-36 (2008)]. Several scenarios can explain the emergence of cis-regulatory elements that govern novel aspects of a gene expression pattern [M. Rebeiz, M. Tsiantis, Curr. Opin. Genet. Dev. 45, 115-123 (2017)]. One scenario, enhancer co-option, holds that a DNA sequence producing an ancestral regulatory activity also becomes the template for a new regulatory activity, sharing regulatory information. While enhancer co-option might fuel morphological diversification, it has rarely been documented [W. J. Glassford et al., Dev. Cell 34, 520-531 (2015)]. Moreover, if two regulatory activities are borne from the same sequence, their modularity, considered a defining feature of enhancers [J. Banerji, L. Olson, W. Schaffner, Cell 33, 729-740 (1983)], might be affected by pleiotropy. Sequence overlap may thereby play a determinant role in enhancer function and evolution. Here, we investigated this problem with two regulatory activities of the Drosophila gene yellow, the novel spot enhancer and the ancestral wing blade enhancer. We used precise and comprehensive quantification of each activity in Drosophila wings to systematically map their sequences along the locus. We show that the spot enhancer has co-opted the sequences of the wing blade enhancer. We also identified a pleiotropic site necessary for DNA accessibility of a shared regulatory region. While the evolutionary steps leading to the derived activity are still unknown, such pleiotropy suggests that enhancer accessibility could be one of the molecular mechanisms seeding evolutionary co-option.

ATAC-seq reveals regional differences in enhancer accessibility during the establishment of spatial coordinates in the Drosophila blastoderm.

Establishment of spatial coordinates during Drosophila embryogenesis relies on differential regulatory activity of axis patterning enhancers. Concentration gradients of activator and repressor transcription factors (TFs) provide positional information to each enhancer, which in turn promotes transcription of a target gene in a specific spatial pattern. However, the interplay between an enhancer regulatory activity and its accessibility as determined by local chromatin organization is not well understood. We profiled chromatin accessibility with ATAC-seq in narrow, genetically tagged domains along the antero-posterior axis in the Drosophila blastoderm. We demonstrate that one-quarter of the accessible genome displays significant regional variation in its ATAC-seq signal immediately after zygotic genome activation. Axis patterning enhancers are enriched among the most variable intervals, and their accessibility changes correlate with their regulatory activity. In an embryonic domain where an enhancer receives a net activating TF input and promotes transcription, it displays elevated accessibility in comparison to a domain where it receives a net repressive input. We propose that differential accessibility is a signature of patterning cis-regulatory elements in the Drosophila blastoderm and discuss potential mechanisms by which accessibility of enhancers may be modulated by activator and repressor TFs.

Developmental Transcriptional Enhancers: A Subtle Interplay between Accessibility and Activity: Considering Quantitative Accessibility Changes between Different Regulatory States of an Enhancer Deconvolutes the Complex Relationship between Accessibility and Activity.

Measurements of open chromatin in specific cell types are widely used to infer the spatiotemporal activity of transcriptional enhancers. How reliable are these predictions? In this review, it is argued that the relationship between the accessibility and activity of an enhancer is insufficiently described by simply considering open versus closed chromatin, or active versus inactive enhancers. Instead, recent studies focusing on the quantitative nature of accessibility signal reveal subtle differences between active enhancers and their different inactive counterparts: the closed silenced state and the accessible primed and repressed states. While the open structure as such is not a specific indicator of enhancer activity, active enhancers display a higher degree of accessibility than the primed and repressed states. Molecular mechanisms that may account for these quantitative differences are discussed. A model that relates molecular events at an enhancer to changes in its activity and accessibility in a developing tissue is also proposed.

Correction: Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines.

[This corrects the article DOI: 10.1371/journal.pbio.1002454.].

Revisiting the developmental and cellular role of the pigmentation gene yellow in Drosophila using a tagged allele.

Pigmentation is a diverse and ecologically relevant trait in insects. Pigment formation has been studied extensively at the genetic and biochemical levels. The temporality of pigment formation during animal development, however, is more elusive. Here, we examine this temporality, focusing on yellow, a gene involved in the formation of black melanin. We generated a protein-tagged yellow allele in the fruit fly Drosophila melanogaster, which allowed us to precisely describe Yellow expression pattern at the tissue and cellular levels throughout development. We found Yellow expressed in the pupal epidermis in patterns prefiguring black pigmentation. We also found Yellow expressed in a few central neurons from the second larval instar to adult stages, including a subset of neurons adjacent to the clock neurons marked by the gene Pdf. We then specifically examined the dynamics of Yellow expression domain and subcellular localization in relationship to pigment formation. In particular, we showed how a late step of re-internalization is regulated by the large low-density lipoprotein receptor-related protein Megalin. Finally we suggest a new function for Yellow in the establishment of sharp pigmentation pattern boundaries, whereby this protein may assume a structural role, anchoring pigment deposits or pigmentation enzymes in the cuticle.

A short guide to insect oviposition: when, where and how to lay an egg.

Egg-laying behavior is one of the most important aspects of female behavior, and has a profound impact on the fitness of a species. As such, it is controlled by several layers of regulation. Here, we review recent advances in our understanding of insect neural circuits that control when, where and how to lay an egg. We also outline outstanding open questions about the control of egg-laying decisions, and speculate on the possible neural underpinnings that can drive the diversification of oviposition behaviors through evolution.

Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines.

The ability to find and consume nutrient-rich diets for successful reproduction and survival is fundamental to animal life. Among the nutrients important for all animals are polyamines, a class of pungent smelling compounds required in numerous cellular and organismic processes. Polyamine deficiency or excess has detrimental effects on health, cognitive function, reproduction, and lifespan. Here, we show that a diet high in polyamine is beneficial and increases reproductive success of flies, and we unravel the sensory mechanisms that attract Drosophila to polyamine-rich food and egg-laying substrates. Using a combination of behavioral genetics and in vivo calcium imaging, we demonstrate that Drosophila uses multisensory detection to find and evaluate polyamines present in overripe and fermenting fruit, their favored feeding and egg-laying substrate. In the olfactory system, two coexpressed ionotropic receptors (IRs), IR76b and IR41a, mediate the long-range attraction to the odor. In the gustatory system, multimodal taste sensation by IR76b receptor and GR66a bitter receptor neurons is used to evaluate quality and valence of the polyamine providing a mechanism for the fly's high attraction to polyamine-rich and sweet decaying fruit. Given their universal and highly conserved biological roles, we propose that the ability to evaluate food for polyamine content may impact health and reproductive success also of other animals including humans.

Body plan innovation in treehoppers through the evolution of an extra wing-like appendage.

Body plans, which characterize the anatomical organization of animal groups of high taxonomic rank, often evolve by the reduction or loss of appendages (limbs in vertebrates and legs and wings in insects, for example). In contrast, the addition of new features is extremely rare and is thought to be heavily constrained, although the nature of the constraints remains elusive. Here we show that the treehopper (Membracidae) 'helmet' is actually an appendage, a wing serial homologue on the first thoracic segment. This innovation in the insect body plan is an unprecedented situation in 250 Myr of insect evolution. We provide evidence suggesting that the helmet arose by escaping the ancestral repression of wing formation imparted by a member of the Hox gene family, which sculpts the number and pattern of appendages along the body axis. Moreover, we propose that the exceptional morphological diversification of the helmet was possible because, in contrast to the wings, it escaped the stringent functional requirements imposed by flight. This example illustrates how complex morphological structures can arise by the expression of ancestral developmental potentials and fuel the morphological diversification of an evolutionary lineage.

Regulatory evolution tuning pigmentation intensity quantitatively in Drosophila.

Quantitative variation in attributes such as color, texture, or stiffness dominates morphological diversification. It results from combinations of alleles at many Mendelian loci. Here, we identify an additional source of quantitative variation among species, continuous evolution in a gene regulatory region. Specifically, we examined the modulation of wing pigmentation in a group of fly species and showed that inter-species variation correlated with the quantitative expression of the pigmentation gene yellow. This variation results from an enhancer of yellow determining darkness through species-specific activity. We mapped the divergent activities between two sister species and found the changes to be broadly distributed along the enhancer. Our results demonstrate that enhancers can act as dials fueling quantitative morphological diversification by modulating trait properties.

On the taxonomic status of the tenebrionoid genera Falsopedilus Pic, 1924 and Hintonosia Báguena Corella, 1948 (Coleoptera: Mycteridae) with descriptions of new species.

We re-evaluate the taxonomic placement of the genus Hintonosia Báguena Corella, 1948, hitherto placed in Aderidae Csíki, 1909. The assessment of key morphological characters based on the type specimen of the type species of this genus, Hintonosia bismacrocephalus Báguena Corella, 1948 (= Hintonosia macrocephalus Pic, 1935, homonym), demonstrated that it does not possess the synapomorphies of the Aderidae. Instead, its clear position in Mycteridae Blanchard, 1845 is uncovered, leading us to the transfer of Hintonosia to the subfamily Eurypinae Thomson, 1860 of Mycteridae. Moreover, Hintonosia was found congeneric with the mycterid genus Falsopedilus Pic, 1924 and the new synonymy is therefore proposed. Additionally, two African species of Falsopedilus are described, a key to, and a checklist of Falsopedilus species are presented.

Increased chromatin accessibility promotes the evolution of a transcriptional silencer in Drosophila.

The loss of discrete morphological traits, the most common evolutionary transition, is typically driven by changes in developmental gene expression. Mutations accumulating in regulatory elements of these genes can disrupt DNA binding sites for transcription factors patterning their spatial expression, or delete entire enhancers. Regulatory elements, however, may be silenced through changes in chromatin accessibility or the emergence of repressive elements. Here, we show that increased chromatin accessibility at the gene yellow, combined with the gain of a repressor site, underlies the loss of a wing spot pigmentation pattern in a Drosophila species. The gain of accessibility of this repressive element is regulated by E93, a transcription factor governing the progress of metamorphosis. This convoluted evolutionary scenario contrasts with the parsimonious mutational paths generally envisioned and often documented for morphological losses. It illustrates how evolutionary changes in chromatin accessibility may directly contribute to morphological diversification.

Odor-regulated oviposition behavior in an ecological specialist.

Colonization of a novel ecological niche can require, or be driven by, evolution of an animal's behaviors promoting their reproductive success. We investigated the evolution and sensory basis of oviposition in Drosophila sechellia, a close relative of Drosophila melanogaster that exhibits extreme specialism for Morinda citrifolia noni fruit. D. sechellia produces fewer eggs than other drosophilids and lays these almost exclusively on noni substrates. We show that visual, textural and social cues do not explain this species-specific preference. By contrast, we find that loss of olfactory input in D. sechellia, but not D. melanogaster, essentially abolishes egg-laying, suggesting that olfaction gates gustatory-driven noni preference. Noni odors are detected by redundant olfactory pathways, but we discover a role for hexanoic acid and the cognate Ionotropic receptor 75b (Ir75b) in odor-evoked oviposition. Through receptor exchange in D. melanogaster, we provide evidence for a causal contribution of odor-tuning changes in Ir75b to the evolution of D. sechellia's oviposition behavior.

Repeated morphological evolution through cis-regulatory changes in a pleiotropic gene.

The independent evolution of morphological similarities is widespread. For simple traits, such as overall body colour, repeated transitions by means of mutations in the same gene may be common. However, for more complex traits, the possible genetic paths may be more numerous; the molecular mechanisms underlying their independent origins and the extent to which they are constrained to follow certain genetic paths are largely unknown. Here we show that a male wing pigmentation pattern involved in courtship display has been gained and lost multiple times in a Drosophila clade. Each of the cases we have analysed (two gains and two losses) involved regulatory changes at the pleiotropic pigmentation gene yellow. Losses involved the parallel inactivation of the same cis-regulatory element (CRE), with changes at a few nucleotides sufficient to account for the functional divergence of one element between two sibling species. Surprisingly, two independent gains of wing spots resulted from the co-option of distinct ancestral CREs. These results demonstrate how the functional diversification of the modular CREs of pleiotropic genes contributes to evolutionary novelty and the independent evolution of morphological similarities.

The genus Syzeton Blackburn, 1891 (Coleoptera, Aderidae) in the United States of America.

The species of Syzeton Blackburn, 1891 (=Zonantes Casey, 1895, syn. nov.) known from the United States are revised. All species formerly placed under Zonantes Casey, 1895 are transferred to Syzeton Blackburn, 1891: S. ouachitanus (Werner, 1990), comb. nov.; S. fasciatus (Melsheimer, 1846), comb. nov.; S. hubbardi (Casey, 1895), comb. nov.; S. nubifer (LeConte, 1878), comb. nov.; S. ater (LeConte, 1875), comb. nov.; S. pallidus (Werner, 1990), comb. nov.; S. subfasciatus (LeConte, 1875), comb. nov.; S. floridanus (Werner, 1990), comb. nov.; S. signatus (Haldeman, 1848), comb. nov.). Additionally, three new species are described herein: Syzeton gruberi n. sp., Syzeton arizonae n. sp., both from Arizona, and Syzeton belovi n. sp., from Texas, Oklahoma and Florida. Finally, Zonantes mississippiensis Werner, 1990 is considered a junior synonym of Syzeton ouachitanus (Werner, 1990), syn. nov.

A standardized nomenclature and atlas of the female terminalia of Drosophila melanogaster.

The model organism Drosophila melanogaster has become a focal system for investigations of rapidly evolving genital morphology as well as the development and functions of insect reproductive structures. To follow up on a previous paper outlining unifying terminology for the structures of the male terminalia in this species, we offer here a detailed description of the female terminalia of D. melanogaster. Informative diagrams and micrographs are presented to provide a comprehensive overview of the external and internal reproductive structures of females. We propose a collection of terms and definitions to standardize the terminology associated with the female terminalia in D. melanogaster and we provide a correspondence table with the terms previously used. Unifying terminology for both males and females in this species will help to facilitate communication between various disciplines, as well as aid in synthesizing research across publications within a discipline that has historically focused principally on male features. Our efforts to refine and standardize the terminology should expand the utility of this important model system for addressing questions related to the development and evolution of animal genitalia, and morphology in general.