Origin and diversification of wings: Insights from a neopteran insect

Winged insects underwent an unparalleled evolutionary radiation, but mechanisms underlying the origin and diversification of wings in basal insects are sparsely known compared with more derived holometabolous insects. In the neopteran species Oncopeltus fasciatus, we manipulated wing specification genes and used RNA-seq to obtain both functional and genomic perspectives. Combined with previous studies, our results suggest the following key steps in wing origin and diversification. First, a set of dorsally derived outgrowths evolved along a number of body segments including the first thoracic segment (T1). Homeotic genes were subsequently co-opted to suppress growth of some dorsal flaps in the thorax and abdomen. In T1 this suppression was accomplished by Sex combs reduced, that when experimentally removed, results in an ectopic T1 flap similar to prothoracic winglets present in fossil hemipteroids and other early insects. Global gene-expression differences in ectopic T1 vs. T2/T3 wings suggest that the transition from flaps to wings required ventrally originating cells, homologous with those in ancestral arthropod gill flaps/epipods, to migrate dorsally and fuse with the dorsal flap tissue thereby bringing new functional gene networks; these presumably enabled the T2/T3 wing’s increased size and functionality. Third, “fused” wings became both the wing blade and surrounding regions of the dorsal thorax cuticle, providing tissue for subsequent modifications including wing folding and the fit of folded wings. Finally, Ultrabithorax was co-opted to uncouple the morphology of T2 and T3 wings and to act as a general modifier of hindwings, which in turn governed the subsequent diversification of lineage-specific wing forms.Some 350 million years ago, the development of insect wings was a seminal event in the evolution of insect body design (1, 2). The ability to fly was critical to insects becoming the most diverse and abundant animal group, and the origin of such novelty has been a focus of intense scientific inquiry for more than a century (3, 4). More recently, through studies of genetic model systems such as Drosophila, the mechanisms of wing morphogenesis have been elucidated (5–12). Still lacking however is a comprehensive understanding of transitional steps connecting the morphology of structures observed in the fossil record with that of the modern-day insects, including wing origins and subsequent diversification.The initial stages of insect wing evolution are missing from the fossil record and it is therefore necessary to use indirect evidence from fossils that postdate the origin and initial radiation of pterygotes (2). Larvae of many of those taxa featured dorsally positioned outgrowths on each of the thoracic and abdominal segments (2, 13), apparently serial homologs (i.e., similar structures likely arising from a common set of developmental mechanisms). Diverse lineages independently lost those dorsal appendages on the abdomen while undergoing parallel modifications of wing-like structures on thoracic (T1–T3) segments. Specifically, the T1 winglets were always much smaller in fossils and apparently lacked hinge articulation whereas T2 (fore-) and T3 (hind-) wings were fully operational in adults, featuring muscles, venation, and size that rendered them capable of flapping flight (14). T1 winglets were subsequently repressed in multiple lineages (15–18) whereas T2 and T3 wings acquired morphology similar to modern day Paleoptera (mayflies and dragonflies) and other extinct paleopterous orders. The transition from Paleoptera, which rest with wings extended from the body, to Neoptera, which rest with wings folded flat against the body, required changes in the hinge mechanism, with many orders also evolving a precise mechanical fit between wing margins and the adjacent body wall of the dorsal thorax. Finally, the radiation of Neoptera encompassed a further divergence between the fore- and hindwings in terms of their shape, size, and texture (5, 19, 20). Together, this set of transitions accounts for major features of the diversity and lineage-specific wing morphologies among fossil and extant taxa.To gain insight into genetic mechanisms governing these transitional steps, we used a direct-developing neopteran species, the milkweed bug (Oncopeltus fasciatus; Hemimetabola, Hemiptera). The phylogenetic placement of Oncopeltus, basal to the more derived holometabola (e.g., flies, beetles, bees, butterflies, and so forth that have a pupal stage), is important because holometabolous appendage development occurs from imaginal discs, which are collections of cells that form and commit to appendage identity early in larval development. Oncopeltus and other hemimetabolous insects lack imaginal discs and acquire adult morphology gradually through a series of nymphal stages, similar to early fossil insects. Hence, examination of wing development in a hemimetabolous insect can help resolve the ancestral versus derived status of developmental traits present in holometabola and in general may provide new evolutionary perspectives.Oncopeltus features brightly colored forewings with a stiff proximal region and a more flexible membranous apex (hemelytra), entirely membranous hind wings, and a well-developed dorsal T2 structure (scutellum) that fits precisely against trailing edges of the folded wings. To examine wing developmental mechanisms underlying these features, we combined a candidate gene approach in which we depleted (via RNA interference, RNAi) the expression of Oncopeltus orthologs of wing specification genes, and a global approach (RNA-seq) (21) that characterized all expressed genes in wild-type T2 and T3 wings, ectopic T1 wings, and wild-type T1 body wall. The results provide independent and expanded insights into the origins and fate of T1 wings, the transition from paleoptera to neoptera, and the eventual diversification of T2 and T3 wing morphology.