The paper describes new domains of venation patterning genes in butterflies and proposes how 14 simplified venation in other insect lineages might have evolved. 15 16 Abstract 17 The mechanism of wing vein differentiation in Drosophila is a classic text-book example of 18 pattern formation using a system of positional-information, yet very little is known about how this 19 mechanism differs in species with a different number of veins and how insect venation patterns 20 evolved. Here, we examine the expression patterns of genes previously implicated in vein 21 differentiation in Drosophila in two butterfly species with more complex venation, the African 22 squinting bush brown Bicyclus anynana and the Asian cabbage white, Pieris canidia. We also test 23 the function of one of these genes, spalt (sal), with CRISPR-Cas9 in B. anynana. We identify 24 both conserved as well as new domains of decapentaplegic (dpp), engrailed (en), invected (inv) 25 and sal gene expression in B. anynana, and propose how the simplified venation in Drosophila 26 might have evolved via loss of dpp and sal gene expression domains, silencing of vein inducing 27 programs at Sal-expression boundaries, and changes in gene expression of vein maintenance 28 genes. 29 30 MAIN TEXT 31 32 45 47 vinegar fly Drosophila melanogaster, where a classic system of positional information takes 48 place. Here, the wing is initially sub-divided into two domains of gene expression, and anterior 49 and posterior compartment, where engrailed (en) and invected (inv) expression are restricted to 50 the posterior compartment (7, 8). A central linear morphogen source of the protein 51 Decapentaplegic (Dpp) is established at the posterior border of the anterior compartment, and 52 genes like spalt (sal) respond to the continuous morphogen gradient in a threshold-like manner, 53 creating sharp boundaries of gene expression that provide precise positioning for the longitudinal 54 veins (9, 10). Veins differentiate along these boundaries, along a parallel axis to the Dpp 55 morphogen source (11, 12). Vein cell identity is later determined by the expression of genes such 56 as rhomboid (rho), downstream of sal (10, 13). Conversely, intervein cells will later express 57 blistered (bls) which suppresses vein development (14, 15). The final vein positions are 58 determined by the cross-regulatory interaction of rho and bls. 59 60The mechanisms underlying venation patterning in other insect lineages have remained poorly 61 understood, and so far, gene expression patterns and functions for the few genes examined seem 62 to be similar to those in Drosophila (16-18).
64Venation patterning in butterflies (order: Lepidoptera) has been examined in a few mutants in 65 connection with alterations of color pattern development (19, 20) and more directly via the 66 expression pattern of a few genes during larval development. Two of the species in which a few 67 of the venation patterning genes have been studied in some detail are the African squinting bush 68 brown butterfly,...