Model for nymphalid eyespot and pierid spot development, and evolutionary hypothesis of spot/eyespot evolution. (A) During the late larval stage, Dll and Notch are expressed in several intervenous stripes (purple) midway between the wing veins in both B. anynana and P. rapae butterflies, but only a sub-set of wing cells express these and several other genes in enlarged foci at the end of those stripes, including sal (dark green; forewing represented here where only two foci differentiate). PSmad (red) is also present along the wing margin; B) In the early stages of pupal development, pSmad (red) is expressed in the future eyespot centers of B. anynana, but not in the spot centers of P. rapae. A gradient of this protein, however, may be present along the proximal-distal axis of the wing, being established from the marginal expression of TGF-β ligands in the late larval wings. This gradient could display a similar range of concentrations along bands spanning the anterior-posterior axis of the wing (red dashed lines); C) Later in pupal development while in B. anynana sal (light green) responds to pSmad levels generated from focal signaling, in P. rapae, sal is responding to particular pSmad levels expressed along the anterior-posterior axis (see text for further explanation of spot pattern). (D) The expression of sal determines where black scales will develop on the adult wing in both species. (E). Phylogenetic tree depicting a member from each lepidopteran family studied. The appearance of novel (non-homologous) features are depicted on the tree. These features can represent a novel phenotype, a novel gene expression pattern, or a novel gene circuit (see text for description). Note that additional information was used to map the origin of the discal-cell eyespots at the base of the tree, rather than in the branch representing the saturniid lineage, which would have been more parsimonious (see text).