Multi-scale dissection of wing transparency in the clearwing butterfly Phanus vitreus.

Optical transparency is rare in terrestrial organisms, and often originates through loss of pigmentation and reduction in scattering. The coloured wings of some butterflies and moths have repeatedly evolved transparency, offering examples of how they function optically and biologically. Because pigments are primarily localized in the scales that cover a colourless wing membrane, transparency has often evolved through the complete loss of scales or radical modification of their shape. Whereas bristle-like scales have been well documented in glasswing butterflies, other scale modifications resulting in transparency remain understudied. The butterfly Phanus vitreus achieves transparency while retaining its scales and exhibiting blue/cyan transparent zones. Here, we investigate the mechanism of wing transparency in P. vitreus by light microscopy, focused ion beam milling, microspectrophotometry and optical modelling. We show that transparency is achieved via loss of pigments and vertical orientation in normal paddle-like scales. These alterations are combined with an anti-reflective nipple array on portions of the wing membrane being more exposed to light. The blueish coloration of the P. vitreus transparent regions is due to the properties of the wing membrane, and local scale nanostructures. We show that scale retention in the transparent patches might be explained by these perpendicular scales having hydrophobic properties.

Antennapedia and optix regulate metallic silver wing scale development and cell shape in Bicyclus anynana butterflies.

Butterfly wing scales can develop intricate cuticular nanostructures that produce silver colors, but the underlying genetic and physical basis of such colors is mostly unexplored. Here, we characterize different types of wild-type silver scales in Bicyclus anynana butterflies and show that the varying thickness of the air layer between two cuticular laminas is most important for producing silvery broadband reflectance. We then address the function of five genes-apterous A, Ultrabithorax, doublesex, Antennapedia, and optix-in silver scale development by examining crispants with either ectopic gains or losses of silver scales. Simultaneous transformations of three parameters-loss of the upper lamina, increased lower lamina thickness, and increased pigmentation-occur when silver scales become brown and vice versa when brown scales become silver. Antennapedia and optix are high-level regulators of different silver scale types and determine cell shape in both sexes. Moreover, Antennapedia is involved in determining ridge and crossrib orientation.

Leg length and bristle density, both necessary for water surface locomotion, are genetically correlated in water striders.

Access to hitherto unexploited ecological opportunities is associated with phenotypic evolution and often results in significant lineage diversification. Yet our understanding of the mechanisms underlying such adaptive traits remains limited. Water striders have been able to exploit the water-air interface, primarily facilitated by changes in the density of hydrophobic bristles and a significant increase in leg length. These two traits are functionally correlated and are both necessary for generating efficient locomotion on the water surface. Whether bristle density and leg length have any cellular or developmental genetic mechanisms in common is unknown. Here, we combine comparative genomics and transcriptomics with functional RNA interference assays to examine the developmental genetic and cellular mechanisms underlying the patterning of the bristles and the legs in Gerris buenoi and Mesovelia mulsanti, two species of water striders. We found that two duplication events in the genes beadex and taxi led to a functional expansion of the paralogs, which affected bristle density and leg length. We also identified genes for which no function in bristle development has been previously described in other insects. Interestingly, most of these genes play a dual role in regulating bristle development and leg length. In addition, these genes play a role in regulating cell division. This result suggests that cell division may be a common mechanism through which these genes can simultaneously regulate leg length and bristle density. We propose that pleiotropy, through which gene function affects the development of multiple traits, may play a prominent role in facilitating access to unexploited ecological opportunities and species diversification.

Repression precedes independent evolutionary gains of a highly specific gene expression pattern.

Highly specific expression patterns can be caused by the overlapping activities of activator and repressor sequences in enhancers. However, few studies illuminate how these sequences evolve in the origin of new enhancers. Here, we show that expression of the bond gene in the semicircular wall epithelium (swe) of the Drosophila melanogaster male ejaculatory bulb (EB) is controlled by an enhancer consisting of an activator region that requires Abdominal-B driving expression in the entire EB and a repressor region that restricts this expression to the EB swe. Although this expression pattern is independently gained in the distantly related Scaptodrosophila lebanonensis and does not require Abdominal-B, we show that functionally similar repressor sequences are present in Scaptodrosophila and also in species that do not express bond in the EB. We suggest that during enhancer evolution, repressor sequences can precede the evolution of activator sequences and may lead to similar but independently evolved expression patterns.

DrosoPhyla: Resources for Drosophilid Phylogeny and Systematics.

The vinegar fly Drosophila melanogaster is a pivotal model for invertebrate development, genetics, physiology, neuroscience, and disease. The whole family Drosophilidae, which contains over 4,400 species, offers a plethora of cases for comparative and evolutionary studies. Despite a long history of phylogenetic inference, many relationships remain unresolved among the genera, subgenera, and species groups in the Drosophilidae. To clarify these relationships, we first developed a set of new genomic markers and assembled a multilocus data set of 17 genes from 704 species of Drosophilidae. We then inferred a species tree with highly supported groups for this family. Additionally, we were able to determine the phylogenetic position of some previously unplaced species. These results establish a new framework for investigating the evolution of traits in fruit flies, as well as valuable resources for systematics.

Phylogeny and evolution of mycophagy in the Zygothrica genus group (Diptera: Drosophilidae).

Despite numerous phylogenetic studies on the family Drosophilidae, relationships among some important lineages are still poorly resolved. An example is the equivocal position of the Zygothrica genus group that is mostly comprised of the mycophagous genera Hirtodrosophila, Mycodrosophila, Paramycodrosophila, and Zygothrica. To fill this gap, we conducted a phylogenetic analysis by assembling a dataset of 24 genes from 92 species, including 42 species of the Zygothrica genus group mainly from the Palearctic and Oriental regions. The resulting tree shows that the Zygothrica genus group is monophyletic and places it as the sister to the genus Dichaetophora, and the clade Zygothrica genus group + Dichaetophora is sister to the Siphlodora + Idiomyia/Scaptomyza clade. Within the Zygothrica genus group, the genera Mycodrosophila and Paramycodrosophila are both recognized as monophyletic, while neither the genus Zygothrica nor Hirtodrosophila is monophyletic. We also used this phylogenetic tree to investigate the evolution of mycophagy by reconstructing ancestral food habit in the Drosophilidae. We found that fungus-feeding habit has been gained independently in two lineages. The most recent common ancestor (MRCA) of the subgenus Drosophila was estimated to have acquired mycophagy by expanding its ancestral feeding niche on fermenting fruits to decayed fungi, while the MRCA of the Zygothrica genus group shifted its niche from fruits to fungi as a specialist probably preferring fresh fruiting bodies.

Cellular and developmental basis of avian structural coloration.

Vivid structural colors in birds are a conspicuous and vital part of their phenotype. They are produced by a rich diversity of integumentary photonic nanostructures in skin and feathers. Unlike pigmentary coloration, whose genetic basis is being elucidated, little is known regarding the pathways underpinning organismal structural coloration. Here, we review available data on the development of avian structural colors. In particular, feather photonic nanostructures are understood to be intracellularly self-assembled by physicochemical forces typically seen in soft colloidal systems. We identify promising avenues for future research that can address current knowledge gaps, which are also highly relevant for the sustainable engineering of advanced bioinspired and biomimetic materials.

The evolution of insect metallothioneins.

Metallothioneins (MTs) are a family of cysteine-rich metal-binding proteins that are important in the chelating and detoxification of toxic heavy metals. Until now, the short length and the low sequence complexity of MTs have hindered the inference of robust phylogenies, hampering the study of their evolution. To address this longstanding question, we applied an iterative BLAST search pipeline that allowed us to build a unique dataset of more than 300 MT sequences in insects. By combining phylogenetics and synteny analysis, we reconstructed the evolutionary history of MTs in insects. We show that the MT content in insects has been shaped by lineage-specific tandem duplications from a single ancestral MT. Strikingly, we also uncovered a sixth MT, MtnF, in the model organism Drosophila melanogaster. MtnF evolves faster than other MTs and is characterized by a non-canonical length and higher cysteine content. Our methodological framework not only paves the way for future studies on heavy metal detoxification but can also allow us to identify other previously unidentified genes and other low complexity genomic features.

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.

Birth-and-Death Evolution of the Fatty Acyl-CoA Reductase (FAR) Gene Family and Diversification of Cuticular Hydrocarbon Synthesis in Drosophila.

The birth-and-death evolutionary model proposes that some members of a multigene family are phylogenetically stable and persist as a single copy over time, whereas other members are phylogenetically unstable and undergo frequent duplication and loss. Functional studies suggest that stable genes are likely to encode essential functions, whereas rapidly evolving genes reflect phenotypic differences in traits that diverge rapidly among species. One such class of rapidly diverging traits are insect cuticular hydrocarbons (CHCs), which play dual roles in chemical communications as short-range recognition pheromones as well as protecting the insect from desiccation. Insect CHCs diverge rapidly between related species leading to ecological adaptation and/or reproductive isolation. Because the CHC and essential fatty acid biosynthetic pathways share common genes, we hypothesized that genes involved in the synthesis of CHCs would be evolutionary unstable, whereas those involved in fatty acid-associated essential functions would be evolutionary stable. To test this hypothesis, we investigated the evolutionary history of the fatty acyl-CoA reductases (FARs) gene family that encodes enzymes in CHC synthesis. We compiled a unique data set of 200 FAR proteins across 12 Drosophila species. We uncovered a broad diversity in FAR content which is generated by gene duplications, subsequent gene losses, and alternative splicing. We also show that FARs expressed in oenocytes and presumably involved in CHC synthesis are more unstable than FARs from other tissues. Taken together, our study provides empirical evidence that a comparative approach investigating the birth-and-death evolution of gene families can identify candidate genes involved in rapidly diverging traits between species.

Actomyosin-Driven Tension at Compartmental Boundaries Orients Cell Division Independently of Cell Geometry In Vivo.

Cell shape is known to influence the plane of cell division. In vitro, mechanical constraints can also orient mitoses; however, in vivo it is not clear whether tension can orient the mitotic spindle directly, because tissue-scale forces can change cell shape. During segmentation of the Drosophila embryo, actomyosin is enriched along compartment boundaries forming supracellular cables that keep cells segregated into distinct compartments. Here, we show that these actomyosin cables orient the planar division of boundary cells perpendicular to the boundaries. This bias overrides the influence of cell shape, when cells are mildly elongated. By decreasing actomyosin cable tension with laser ablation or, conversely, ectopically increasing tension with laser wounding, we demonstrate that local tension is necessary and sufficient to orient mitoses in vivo. This involves capture of the spindle pole by the actomyosin cortex. These findings highlight the importance of actomyosin-mediated tension in spindle orientation in vivo.

The achaete-scute complex contains a single gene that controls bristle development in the semi-aquatic bugs.

The semi-aquatic bugs (Heteroptera, Gerromorpha) conquered water surfaces worldwide and diversified to occupy puddles, ponds, streams, lakes, mangroves and even oceans. Critical to this lifestyle is the evolution of sets of hairs that allow these insects to maintain their body weight on the water surface and protect the animals against wetting and drowning. In addition, the legs of these insects are equipped with various grooming combs that are important for cleaning and tidying the hair layers for optimal functional efficiency. Here we show that the hairs covering the legs of water striders represent innervated bristles. Genomic and transcriptomic analyses revealed that in water striders the achaete-scute complex, known to control bristle development in flies, contains only the achaete-scute homologue (ASH) gene owing to the loss of the gene asense. Using RNA interference, we show that ASH plays a pivotal role in the development of both bristles and grooming combs in water striders. Our data suggest that the ASH locus may have contributed to the adaptation to water surface lifestyle through shaping the hydrophobic bristles that prevent water striders from wetting and allow them to exploit water surface tension.

The genome of the water strider Gerris buenoi reveals expansions of gene repertoires associated with adaptations to life on the water.

BACKGROUND: Having conquered water surfaces worldwide, the semi-aquatic bugs occupy ponds, streams, lakes, mangroves, and even open oceans. The diversity of this group has inspired a range of scientific studies from ecology and evolution to developmental genetics and hydrodynamics of fluid locomotion. However, the lack of a representative water strider genome hinders our ability to more thoroughly investigate the molecular mechanisms underlying the processes of adaptation and diversification within this group. RESULTS: Here we report the sequencing and manual annotation of the Gerris buenoi (G. buenoi) genome; the first water strider genome to be sequenced thus far. The size of the G. buenoi genome is approximately 1,000 Mb, and this sequencing effort has recovered 20,949 predicted protein-coding genes. Manual annotation uncovered a number of local (tandem and proximal) gene duplications and expansions of gene families known for their importance in a variety of processes associated with morphological and physiological adaptations to a water surface lifestyle. These expansions may affect key processes associated with growth, vision, desiccation resistance, detoxification, olfaction and epigenetic regulation. Strikingly, the G. buenoi genome contains three insulin receptors, suggesting key changes in the rewiring and function of the insulin pathway. Other genomic changes affecting with opsin genes may be associated with wavelength sensitivity shifts in opsins, which is likely to be key in facilitating specific adaptations in vision for diverse water habitats. CONCLUSIONS: Our findings suggest that local gene duplications might have played an important role during the evolution of water striders. Along with these findings, the sequencing of the G. buenoi genome now provides us the opportunity to pursue exciting research opportunities to further understand the genomic underpinnings of traits associated with the extreme body plan and life history of water striders.

Evolution of the YABBY gene family in seed plants.

Members of the YABBY gene family of transcription factors in angiosperms have been shown to be involved in the initiation of outgrowth of the lamina, the maintenance of polarity, and establishment of the leaf margin. Although most of the dorsal-ventral polarity genes in seed plants have homologs in non-spermatophyte lineages, the presence of YABBY genes is restricted to seed plants. To gain insight into the origin and diversification of this gene family, we reconstructed the evolutionary history of YABBY gene lineages in seed plants. Our findings suggest that either one or two YABBY genes were present in the last common ancestor of extant seed plants. We also examined the expression of YABBY genes in the gymnosperms Ephedra distachya (Gnetales), Ginkgo biloba (Ginkgoales), and Pseudotsuga menziesii (Coniferales). Our data indicate that some YABBY genes are expressed in a polar (abaxial) manner in leaves and female cones in gymnosperms. We propose that YABBY genes already acted as polarity genes in the last common ancestor of extant seed plants.

Evolution of the ARF gene family in land plants: old domains, new tricks.

Auxin response factors (ARF) are key players in plant development. They mediate the cellular response to the plant hormone auxin by activating or repressing the expression of downstream developmental genes. The pivotal activation function of ARF proteins is enabled by their four-domain architecture, which includes both DNA-binding and protein dimerization motifs. To determine the evolutionary origin of this characteristic architecture, we built a comprehensive data set of 224 ARF-related protein sequences that represents all major living divisions of land plants, except hornworts. We found that ARFs are split into three subfamilies that could be traced back to the origin of the land plants. We also show that repeated events of extensive gene duplication contributed to the expansion of those three original subfamilies. Further examination of our data set uncovered a broad diversity in the structure of ARF transcripts and allowed us to identify an additional conserved motif in ARF proteins. We found that additional structural diversity in ARF proteins is mainly generated by two mechanisms: genomic truncation and alternative splicing. We propose that the loss of domains from the canonical, four-domain ARF structure has promoted functional shifts within the ARF family by disrupting either dimerization or DNA-binding capabilities. For instance, the loss of dimerization domains in some ARFs from moss and spikemoss genomes leads to proteins that are reminiscent of Aux/IAA proteins, possibly providing a clue on the evolution of these modulators of ARF function. We also assessed the functional impact of alternative splicing in the case of ARF4, for which we have identified a novel isoform in Arabidopsis thaliana. Genetic analysis showed that these two transcripts exhibit markedly different developmental roles in A. thaliana. Gene duplications, domain rearrangement, and post-transcriptional regulation have thus enabled a subtle control of auxin signaling through ARF proteins that may have contributed to the critical importance of these regulators in plant development and evolution.

Auxology: when auxin meets plant evo-devo.

Auxin is implicated throughout plant growth and development. Although the effects of this plant hormone have been recognized for more than a century, it is only in the past two decades that light has been shed on the molecular mechanisms that regulate auxin homeostasis, signaling, transport, crosstalk with other hormonal pathways as well as its roles in plant development. These discoveries established a molecular framework to study the role of auxin in land plant evolution. Here, we review recent advances in auxin biology and their implications in both micro- and macro-evolution of plant morphology. By analogy to the term 'hoxology', which refers to the critical role of HOX genes in metazoan evolution, we propose to introduce the term 'auxology' to take into account the crucial role of auxin in plant evo-devo.

The Selaginella genome identifies genetic changes associated with the evolution of vascular plants.

Vascular plants appeared ~410 million years ago, then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes. We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first nonseed vascular plant genome reported. By comparing gene content in evolutionarily diverse taxa, we found that the transition from a gametophyte- to a sporophyte-dominated life cycle required far fewer new genes than the transition from a nonseed vascular to a flowering plant, whereas secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in posttranscriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the trans-acting small interfering RNA pathway, and extensive RNA editing of organellar genes.

Cabomba as a model for studies of early angiosperm evolution.

BACKGROUND: The angiosperms, or flowering plants, diversified in the Cretaceous to dominate almost all terrestrial environments. Molecular phylogenetic studies indicate that the orders Amborellales, Nymphaeales and Austrobaileyales, collectively termed the ANA grade, diverged as separate lineages from a remaining angiosperm clade at a very early stage in flowering plant evolution. By comparing these early diverging lineages, it is possible to infer the possible morphology and ecology of the last common ancestor of the extant angiosperms, and this analysis can now be extended to try to deduce the developmental mechanisms that were present in early flowering plants. However, not all species in the ANA grade form convenient molecular-genetic models. SCOPE: The present study reviews the genus Cabomba (Nymphaeales), which shows a range of features that make it potentially useful as a genetic model. We focus on characters that have probably been conserved since the last common ancestor of the extant flowering plants. To facilitate the use of Cabomba as a molecular model, we describe methods for its cultivation to flowering in the laboratory, a novel Cabomba flower expressed sequence tag database, a well-adapted in situ hybridization protocol and a measurement of the nuclear genome size of C. caroliniana. We discuss the features required for species to become tractable models, and discuss the relative merits of Cabomba and other ANA-grade angiosperms in molecular-genetic studies aimed at understanding the origin of the flowering plants.

Insights from ANA-grade angiosperms into the early evolution of CUP-SHAPED COTYLEDON genes.

BACKGROUND AND AIMS: The closely related NAC family genes NO APICAL MERISTEM (NAM) and CUP-SHAPED COTYLEDON3 (CUC3) regulate the formation of boundaries within and between plant organs. NAM is post-transcriptionally regulated by miR164, whereas CUC3 is not. To gain insight into the evolution of NAM and CUC3 in the angiosperms, we analysed orthologous genes in early-diverging ANA-grade angiosperms and gymnosperms. METHODS: We obtained NAM- and CUC3-like sequences from diverse angiosperms and gymnosperms by a combination of reverse transcriptase PCR, cDNA library screening and database searching, and then investigated their phylogenetic relationships by performing maximum-likelihood reconstructions. We also studied the spatial expression patterns of NAM, CUC3 and MIR164 orthologues in female reproductive tissues of Amborella trichopoda, the probable sister to all other flowering plants. KEY RESULTS: Separate NAM and CUC3 orthologues were found in early-diverging angiosperms, but not in gymnosperms, which contained putative orthologues of the entire NAM + CUC3 clade that possessed sites of regulation by miR164. Multiple paralogues of NAM or CUC3 genes were noted in certain taxa, including Brassicaceae. Expression of NAM, CUC3 and MIR164 orthologues from Am. trichopoda was found to co-localize in ovules at the developmental boundary between the chalaza and nucellus. CONCLUSIONS: The NAM and CUC3 lineages were generated by duplication, and CUC3 was subsequently lost regulation by miR164, prior to the last common ancestor of the extant angiosperms. However, the paralogous NAM clade genes CUC1 and CUC2 were generated by a more recent duplication, near the base of Brassicaceae. The function of NAM and CUC3 in defining a developmental boundary in the ovule appears to have been conserved since the last common ancestor of the flowering plants, as does the post-transcriptional regulation in ovule tissues of NAM by miR164.