WntA expression and wing transcriptomics illuminate the evolution of stripe patterns in skipper butterflies

Abstract

Skippers (Hesperiidae) form a distinct lineage of butterflies where the developmental mechanisms of color patterning have seldom been studied. The wing patterns of skippers often consist of median stripes, and classic comparative morphology studies suggest these elements are homologous to the Central Symmetry System (CSS) found in nymphalid butterflies. Consistent with this hypothesis, we show here that expression of the signaling ligand gene WntA, a marker of the CSS in nymphalids, prefigures the position of the main wing patterns of the silver-spotted skipper Epargyreus clarus. Notably, WntA expression is associated with different color outputs in the forewing vs. hindwing, validating a theory of pattern homology from the mid-twentieth century. To further gain insights into the genes associated with this patterning process, we generated an annotated genome for E. clarus and performed an RNAseq study profiling gene expression along the proximo-distal (P-D) axis of early pupal wings. These data suggest that the transcription factor genes lobe, u-shaped, and odd-paired are expressed in restricted P-D sections of the wing similarly to WntA, indicating they may participate in the patterning of the CSS. In addition, inverted expression patterns of dachsous and four-jointed, as well as the expression of transcription factors with roles in specifying proximal (homothorax, tiptop/teashirt) and distal (vestigial, scalloped) section of ths Drosophila wing disk, suggest a deep conservation of wing P-D patterning process between Diptera and Lepidoptera. This work highlights the developmental homology between the CSS of Hesperiidae and Nymphalidae, and enriches our knowledge of patterning mechanisms in lepidopteran wings. Summary StatementThis study reveals how skipper butterfly wing stripes share deep developmental origins with other butterflies, offering fresh insight into wing pattern evolution.