Chironomus riparius (Diptera) genome sequencing reveals the impact of minisatellite transposable elements on population divergence.

Active transposable elements (TEs) may result in divergent genomic insertion and abundance patterns among conspecific populations. Upon secondary contact, such divergent genetic backgrounds can theoretically give rise to classical Dobzhansky-Muller incompatibilities (DMI), thus contributing to the evolution of endogenous genetic barriers and eventually causing population divergence. We investigated differential TE abundance among conspecific populations of the nonbiting midge Chironomus riparius and evaluated their potential role in causing endogenous genetic incompatibilities between these populations. We focussed on a Chironomus-specific TE, the minisatellite-like Cla-element, whose activity is associated with speciation in the genus. Using a newly generated and annotated draft genome for a genomic study with five natural C. riparius populations, we found highly population-specific TE insertion patterns with many private insertions. A significant correlation of the pairwise FST estimated from genomewide single-nucleotide polymorphisms (SNPs) and the FST estimated from TEs is consistent with drift as the major force driving TE population differentiation. However, the significantly higher Cla-element FST level due to a high proportion of differentially fixed Cla-element insertions also indicates selection against segregating (i.e. heterozygous) insertions. With reciprocal crossing experiments and fluorescent in situ hybridization of Cla-elements to polytene chromosomes, we documented phenotypic effects on female fertility and chromosomal mispairings. We propose that the inferred negative selection on heterozygous Cla-element insertions may cause endogenous genetic barriers and therefore acts as DMI among C. riparius populations. The intrinsic genomic turnover exerted by TEs may thus have a direct impact on population divergence that is operationally different from drift and local adaptation.

BMP-dependent serosa and amnion specification in the scuttle fly Megaselia abdita.

Bone morphogenetic protein (BMP) signaling is an essential factor in dorsoventral patterning of animal embryos but how BMP signaling evolved with fundamental changes in dorsoventral tissue differentiation is unclear. Flies experienced an evolutionary reduction of extra-embryonic tissue types from two (amniotic and serosal tissue) to one (amnionserosal tissue). BMP-dependent amnioserosa specification has been studied in Drosophila melanogaster. However, the mechanisms of serosal and amniotic tissue specification in less diverged flies remain unknown. To better understand potential evolutionary links between BMP signaling and extra-embryonic tissue specification, we examined the activity profile and function of BMP signaling in serosa and amnion patterning of the scuttle fly Megaselia abdita (Phoridae) and compared the BMP activity profiles between M. abdita and D. melanogaster. In blastoderm embryos of both species, BMP activity peaked at the dorsal midline. However, at the beginning of gastrulation, peak BMP activity in M. abdita shifted towards prospective amnion tissue. This transition correlated with the first signs of amnion differentiation laterally adjacent to the serosa anlage. Marker-assisted analysis of six BMP signaling components (dpp, gbb, scw, tkv, sax, sog) by RNA interference revealed that both serosa and amnion specification of M. abdita are dependent on BMP activity. Conversely, BMP gain-of-function experiments caused sharpened expression boundaries of extra-embryonic target genes indicative of positive feedback. We propose that changes in the BMP activity profile at the beginning of gastrulation might have contributed to the reduction of extra-embryonic tissue types during the radiation of cyclorrhaphan flies.

Episodic radiations in the fly tree of life.

Flies are one of four superradiations of insects (along with beetles, wasps, and moths) that account for the majority of animal life on Earth. Diptera includes species known for their ubiquity (Musca domestica house fly), their role as pests (Anopheles gambiae malaria mosquito), and their value as model organisms across the biological sciences (Drosophila melanogaster). A resolved phylogeny for flies provides a framework for genomic, developmental, and evolutionary studies by facilitating comparisons across model organisms, yet recent research has suggested that fly relationships have been obscured by multiple episodes of rapid diversification. We provide a phylogenomic estimate of fly relationships based on molecules and morphology from 149 of 157 families, including 30 kb from 14 nuclear loci and complete mitochondrial genomes combined with 371 morphological characters. Multiple analyses show support for traditional groups (Brachycera, Cyclorrhapha, and Schizophora) and corroborate contentious findings, such as the anomalous Deuterophlebiidae as the sister group to all remaining Diptera. Our findings reveal that the closest relatives of the Drosophilidae are highly modified parasites (including the wingless Braulidae) of bees and other insects. Furthermore, we use micro-RNAs to resolve a node with implications for the evolution of embryonic development in Diptera. We demonstrate that flies experienced three episodes of rapid radiation--lower Diptera (220 Ma), lower Brachycera (180 Ma), and Schizophora (65 Ma)--and a number of life history transitions to hematophagy, phytophagy, and parasitism in the history of fly evolution over 260 million y.

Maternal activation of gap genes in the hover fly Episyrphus.

The metameric organization of the insect body plan is initiated with the activation of gap genes, a set of transcription-factor-encoding genes that are zygotically expressed in broad and partially overlapping domains along the anteroposterior (AP) axis of the early embryo. The spatial pattern of gap gene expression domains along the AP axis is generally conserved, but the maternal genes that regulate their expression are not. Building on the comprehensive knowledge of maternal gap gene activation in Drosophila, we used loss- and gain-of-function experiments in the hover fly Episyrphus balteatus (Syrphidae) to address the question of how the maternal regulation of gap genes evolved. We find that, in Episyrphus, a highly diverged bicoid ortholog is solely responsible for the AP polarity of the embryo. Episyrphus bicoid represses anterior zygotic expression of caudal and activates the anterior and central gap genes orthodenticle, hunchback and Krüppel. In bicoid-deficient Episyrphus embryos, nanos is insufficient to generate morphological asymmetry along the AP axis. Furthermore, we find that torso transiently regulates anterior repression of caudal and is required for the activation of orthodenticle, whereas all posterior gap gene domains of knirps, giant, hunchback, tailless and huckebein depend on caudal. We conclude that all maternal coordinate genes have altered their specific functions during the radiation of higher flies (Cyclorrhapha).

Twenty-seven ZAD-ZNF genes of Drosophila melanogaster are orthologous to the embryo polarity determining mosquito gene cucoid.

The C2H2 zinc finger gene cucoid establishes anterior-posterior (AP) polarity in the early embryo of culicine mosquitoes. This gene is unrelated to genes that establish embryo polarity in other fly species (Diptera), such as the homeobox gene bicoid, which serves this function in the traditional model organism Drosophila melanogaster. The cucoid gene is a conserved single copy gene across lower dipterans but nothing is known about its function in other species, and its evolution in higher dipterans, including Drosophila, is unresolved. We found that cucoid is a member of the ZAD-containing C2H2 zinc finger (ZAD-ZNF) gene family and is orthologous to 27 of the 91 members of this family in D. melanogaster, including M1BP, ranshi, ouib, nom, zaf1, odj, Nnk, trem, Zif, and eighteen uncharacterized genes. Available knowledge of the functions of cucoid orthologs in Drosophila melanogaster suggest that the progenitor of this lineage specific expansion may have played a role in regulating chromatin. We also describe many aspects of the gene duplication history of cucoid in the brachyceran lineage of D. melanogaster, thereby providing a framework for predicting potential redundancies among these genes in D. melanogaster.

The generation of variation and the developmental basis for evolutionary novelty.

Organisms exhibit an incredible diversity of form, a fact that makes the evolution of novelty seemingly self-evident. However, despite the "obvious" case for novelty, defining this concept in evolutionary terms is highly problematic, so much so that some have suggested discarding it altogether. Approaches to this problem tend to take either an adaptation- or development-based perspective, but we argue here that an exclusive focus on either of these misses the original intent of the novelty concept and undermines its practical utility. We propose instead that for a feature to be novel, it must have evolved both by a transition between adaptive peaks on the fitness landscape and that this transition must have overcome a previous developmental constraint. This definition focuses novelty on the explanation of apparently difficult or low-probability evolutionary transitions and highlights how the integration of developmental and functional considerations are necessary to evolutionary explanation. It further reinforces that novelty is a central concern not just of evolutionary developmental biology (i.e., "evo-devo") but of evolutionary biology more generally. We explore this definition of novelty in light of four examples that range from the obvious to subtle.

Evolution and loss of ß-catenin and TCF-dependent axis specification in insects.

Mechanisms and evolution of primary axis specification in insects are discussed in the context of the roles of ß-catenin and TCF in polarizing metazoan embryos. Three hypotheses are presented. First, insects with sequential segmentation and posterior growth use cell-autonomous mechanisms for establishing embryo polarity via the nuclear ratio of ß-catenin and TCF. Second, TCF homologs establish competence for anterior specification. Third, the evolution of simultaneous segmentation mechanisms, also known as long-germ development, resulted in primary axis specification mechanisms that are independent of ß-catenin but reliant on TCF, a condition that preceded the frequent replacement of anterior determinants in long germ insects.

How two extraembryonic epithelia became one: serosa and amnion features and functions of Drosophila's amnioserosa.

The conservation of gene networks that specify and differentiate distinct tissues has long been a subject of great interest to evolutionary developmental biologists, but the question of how pre-existing tissue-specific developmental trajectories merge is rarely asked. During the radiation of flies, two extraembryonic epithelia, known as serosa and amnion, evolved into one, called amnioserosa. This unique extraembryonic epithelium is found in fly species of the group Schizophora, including the genetic model organism Drosophila melanogaster, and has been studied in depth. Close relatives of this group develop a serosa and a rudimentary amnion. The scuttle fly Megaselia abdita has emerged as an excellent model organism to study this extraembryonic tissue organization. In this review, development and functions of the extraembryonic tissue complements of Drosophila and Megaselia are compared. It is concluded that the amnioserosa combines cells, genetic pathway components and functions that were previously associated either with serosa development or amnion development. The composite developmental trajectory of the amnioserosa raises the question of whether merging tissue-specific gene networks is a common evolutionary process. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Postgastrular zen expression is required to develop distinct amniotic and serosal epithelia in the scuttle fly Megaselia.

The amnioserosa is an extraembryonic epithelium that evolved in higher cyclorrhaphan flies from distinct serosal and amniotic epithelia. The underlying genetic mechanism of this evolutionary transition is unknown. Amnioserosa development of Drosophila correlates with novel expression characteristics of the homeobox gene zerknüllt (zen), including a broad zen expression domain in the syncytial blastoderm and the complete absence of postgastrular zen expression. Here we examine the functional significance of these features by altering the activity profile of zen in Megaselia (a lower cyclorrhaphan fly with distinct serosal and amniotic epithelia) and Drosophila, and by examining in Megaselia the function of u-shaped group (ush-group) genes, which in Drosophila maintain the amnioserosa after gastrulation when zen is no longer expressed. In Megaselia, loss of postgastrular zen expression abrogates serosa development but allows amnion development. Ectopic expression of zen in early Megaselia embryos allows serosa formation but perturbs amnion development. Megaselia homologues of u-shaped group genes are not essential for serosa formation but mediate germband retraction and dorsal closure. Finally, ectopic postgastrular zen expression in Drosophila causes an enlargement of amnioserosa cells and interferes with the morphogenetic functions of the amnioserosa. Our results suggest that the origin of the amnioserosa involved the loss of postgastrular zen expression from extraembryonic tissue, that the early broad expression domain of Drosophila zen evolved afterwards, and that the ush-group genes ancestrally played a role in morphogenetic functions of the amnion.

BMP signaling components in embryonic transcriptomes of the hover fly Episyrphus balteatus (Syrphidae).

BACKGROUND: In animals, signaling of Bone Morphogenetic Proteins (BMPs) is essential for dorsoventral (DV) patterning of the embryo, but how BMP signaling evolved with changes in embryonic DV differentiation is largely unclear. Based on the extensive knowledge of BMP signaling in Drosophila melanogaster, the morphological diversity of extraembryonic tissues in different fly species provides a comparative system to address this question. The closest relatives of D. melanogaster with clearly distinct DV differentiation are hover flies (Diptera: Syrphidae). The syrphid Episyrphus balteatus is a commercial bio-agent against aphids and has been established as a model organism for developmental studies and chemical ecology. The dorsal blastoderm of E. balteatus gives rise to two extraembryonic tissues (serosa and amnion), whereas in D. melanogaster, the dorsal blastoderm differentiates into a single extraembryonic epithelium (amnioserosa). Recent studies indicate that several BMP signaling components of D. melanogaster, including the BMP ligand Screw (Scw) and other extracellular regulators, evolved in the dipteran lineage through gene duplication and functional divergence. These findings raise the question of whether the complement of BMP signaling components changed with the origin of the amnioserosa. RESULTS: To search for BMP signaling components in E. balteatus, we generated and analyzed transcriptomes of freshly laid eggs (0-30 minutes) and late blastoderm to early germband extension stages (3-6 hours) using Roche/454 sequencing. We identified putative E. balteatus orthologues of 43% of all annotated D. melanogaster genes, including the genes of all BMP ligands and other BMP signaling components. CONCLUSION: The diversification of several BMP signaling components in the dipteran linage of D. melanogaster preceded the origin of the amnioserosa.[Transcriptome sequence data from this study have been deposited at the NCBI Sequence Read Archive (SRP005289); individually assembled sequences have been deposited at GenBank (JN006969-JN006986).].

Evolutionary origin of the amnioserosa in cyclorrhaphan flies correlates with spatial and temporal expression changes of zen.

Higher cyclorrhaphan flies including Drosophila develop a single extraembryonic epithelium (amnioserosa), which closes the germband dorsally. In most other insects two extraembryonic epithelia, serosa and amnion, line the inner eggshell and the ventral germband, respectively. How the two extraembryonic epithelia evolved into one is unclear. Recent studies have shown that, in the flour beetle Tribolium and in the milkweed bug Oncopeltus, the homeobox gene zerknüllt (zen) controls the fusion of the amnion with the serosa before dorsal closure. To understand the origin of the amnioserosa in evolution, we examined the expression and function of zen in the extraembryonic tissue of lower Cyclorrhapha. We show that Megaselia abdita (Phoridae) and Episyrphus balteatus (Syrphidae) develop a serosa and a dorsal amnion, suggesting that a dorsal amnion preceded the origin of the amnioserosa in evolution. Using Krüppel (Kr) and pannier (pnr) homologues of Megaselia as markers for serosal and amniotic tissue, respectively, we show that after zen RNAi all extraembryonic tissue becomes indistinguishable from amniotic cells, like in Tribolium but unlike in Drosophila, in which zen controls all aspects of extraembryonic development. Compared with Megaselia and Episyrphus, zen expression in Drosophila is extended to cells that form the amnion in lower Cyclorrhapha and is down-regulated at the developmental stage, when serosa cells in lower Cyclorrhapha begin to expand. These expression differences between species with distinct extraembryonic tissue organizations and the conserved requirement of zen for serosa development suggest that the origin of an amnioserosa-like epithelium was accompanied by expression changes of zen.

Hox3/zen and the evolution of extraembryonic epithelia in insects.

Insects have undergone dramatic evolutionary changes in extraembryonic development, which correlate with changes in the expression of the class-3 Hox gene zen. Here, we review the evolution of this gene in insects and point out how changes in zen expression may have affected extraembryonic development at the morphological and the genetic level.

Extremely small genomes in two unrelated dipteran insects with shared early developmental traits.

We discovered extremely small genomes (1C ~100 Mb) in the dipteran insects Coboldia fuscipes (Scatopsidae) and Psychoda cinerea (Psychodidae). The small genomes of these species cannot be explained by a fast developmental rate, which has been shown to correlate with small genome sizes in animals and plants but might accommodate the combined effects of other developmental traits, including small egg size, thin blastoderm layer, and long-germ development.

Embryo polarity in moth flies and mosquitoes relies on distinct old genes with localized transcript isoforms.

Unrelated genes establish head-to-tail polarity in embryos of different fly species, raising the question of how they evolve this function. We show that in moth flies (Clogmia, Lutzomyia), a maternal transcript isoform of odd-paired (Zic) is localized in the anterior egg and adopted the role of anterior determinant without essential protein change. Additionally, Clogmia lost maternal germ plasm, which contributes to embryo polarity in fruit flies (Drosophila). In culicine (Culex, Aedes) and anopheline mosquitoes (Anopheles), embryo polarity rests on a previously unnamed zinc finger gene (cucoid), or pangolin (dTcf), respectively. These genes also localize an alternative transcript isoform at the anterior egg pole. Basal-branching crane flies (Nephrotoma) also enrich maternal pangolin transcript at the anterior egg pole, suggesting that pangolin functioned as ancestral axis determinant in flies. In conclusion, flies evolved an unexpected diversity of anterior determinants, and alternative transcript isoforms with distinct expression can adopt fundamentally distinct developmental roles.

Insect serosa: a head line in comparative developmental genetics.

A recent study reveals specific functions of Hox3/zerknüllt (zen) in the extraembryonic and embryonic primordia of the red flour beetle, Tribolium castaneum. The results shed light on the functional evolution of bicoid, a zen paralogue of higher flies, which determines anterior body parts.

Bicoid occurrence and Bicoid-dependent hunchback regulation in lower cyclorrhaphan flies.

The homeobox gene bicoid functions as an anterior pattern organizer of the Drosophila embryo, but other than in higher flies (Cyclorrhapha), bicoid orthologues appear to be absent from insect genomes. In Drosophila, bicoid is expressed in an anterior-to-posterior protein gradient and regulates spatially restricted expression domains of segmentation genes in a concentration-dependent manner. hunchback, a direct transcriptional target of Bicoid, complements the "morphogen" activity of Bicoid. hunchback is activated by Bicoid throughout the anterior half of the blastoderm and a Bicoid-binding cis-regulatory element has been identified immediately upstream of the proximal hunchback promoter P2 of Drosophila and other higher Cyclorrhapha (Schizophora). bicoid and Bicoid-dependent hunchback regulation are thought to have originated during or before the radiation of extant Cyclorrhapha, although the precise occurrence of these traits in lower Cyclorrhapha remains unknown. Previously, we have described a bicoid orthologue in Megaselia, a species of the lower cyclorrhaphan family Phoridae. Here, we report the occurrence of bicoid in two additional lower cyclorrhaphan families, Lonchopteridae and Platypezidae. We show that Megaselia Bicoid is required for anterior expression of Megaselia hunchback, and binds upstream of its P2 promoter. Furthermore, we report the expression of lacZ reporter constructs under the control of hunchback regulatory sequences from a range of lower cyclorrhaphan and non-cyclorrhaphan flies in transgenic Drosophila embryos. Our results are consistent with a cyclorrhaphan origin of bicoid and suggest that a Bicoid-binding enhancer upstream of the hunchback P2 promoter evolved at the latest in the last common ancestor of Megaselia and Schizophora.

Ancient mechanisms for the evolution of the bicoid homeodomain's function in fly development.

The ancient mechanisms that caused developmental gene regulatory networks to diversify among distantly related taxa are not well understood. Here we use ancestral protein reconstruction, biochemical experiments, and developmental assays of transgenic animals carrying reconstructed ancestral genes to investigate how the transcription factor Bicoid (Bcd) evolved its central role in anterior-posterior patterning in flies. We show that most of Bcd's derived functions are attributable to evolutionary changes within its homeodomain (HD) during a phylogenetic interval >140 million years ago. A single substitution from this period (Q50K) accounts almost entirely for the evolution of Bcd's derived DNA specificity in vitro. In transgenic embryos expressing the reconstructed ancestral HD, however, Q50K confers activation of only a few of Bcd's transcriptional targets and yields a very partial rescue of anterior development. Adding a second historical substitution (M54R) confers regulation of additional Bcd targets and further rescues anterior development. These results indicate that two epistatically interacting mutations played a major role in the evolution of Bcd's controlling regulatory role in early development. They also show how ancestral sequence reconstruction can be combined with in vivo characterization of transgenic animals to illuminate the historical mechanisms of developmental evolution.

Emerging developmental genetic model systems in holometabolous insects.

The number of insect species that are amenable to functional genetic studies is growing rapidly and provides many new research opportunities in developmental and evolutionary biology. The holometabolous insects represent a disproportionate percentage of animal diversity and are thus well positioned to provide model species for a wide variety of developmental processes. Here we discuss emerging holometabolous models, and review some recent breakthroughs. For example, flies and midges were found to use structurally unrelated long-range pattern organizers, butterflies and moths revealed extensive pattern formation during oogenesis, new imaging possibilities in the flour beetle Tribolium castaneum showed how embryos break free of their extraembryonic membranes, and the complex genetics governing interspecies difference in head shape were revealed in Nasonia wasps.

Morphogenetic functions of extraembryonic membranes in insects.

Morphogenetic functions of the amnioserosa, the serosa, the amnion, and the yolk sac are reviewed on the basis of recent studies in flies (Drosophila, Megaselia), beetles (Tribolium), and hemipteran bugs (Oncopeltus). Three hypotheses are presented. First, it is suggested that the amnioserosa of Drosophila and the dorsal amnion of other fly species function in a similar manner. Second, it is proposed that in many species with an amniotic cavity, the amnion determines the site of serosa rupture, which, through interactions between the serosa and the amnion, enables the embryo to break free from the amniotic cavity and to close its backside. Finally, it is concluded that the yolk sac is likely an important player in insect morphogenesis.