Understanding how novel complex traits originate involves investigating the time of origin of the trait, as well as the origin of its underlying gene regulatory network in a broad comparative phylogenetic framework. The eyespot of nymphalid butterflies has served as an example of a novel complex trait, as multiple genes are expressed during eyespot development. Yet the origins of eyespots remain unknown. Using a dataset of more than 400 images of butterflies with a known phylogeny and gene expression data for five eyespot-associated genes from over twenty species, we tested origin hypotheses for both eyespots and eyespot-associated genes. We show that eyespots evolved once within the family Nymphalidae, approximately 90 million years ago, concurrent with expression of at least three genes associated with early eyespot development. We also show multiple losses of expression of most genes from this early three-gene cluster, without corresponding losses of eyespots. We propose that complex traits, such as eyespots, may have originated via co-option of a large pre-existing complex gene regulatory network that was subsequently streamlined of genes not required to fulfill its novel developmental function.

This article reviews the latest developments in our understanding of the origin, development, and evolution of nymphalid butterfly eyespots. Recent contributions to this field include insights into the evolutionary and developmental origin of eyespots and their ancestral deployment on the wing, the evolution of eyespot number and eyespot sexual dimorphism, and the identification of genes affecting eyespot development and black pigmentation. I also compare features of old and more recently proposed models of eyespot development and propose a schematic for the genetic regulatory architecture of eyespots. Using this schematic I propose two hypotheses for why we observe limits to morphological diversity across these serially homologous traits.

<h4>Background</h4>One hypothesis surrounding the origin of novel traits is that they originate from the co-option of pre-existing genes or larger gene regulatory networks into novel developmental contexts. Insights into a trait's evolutionary origins can, thus, be gained via identification of the genes underlying trait development, and exploring whether those genes also function in other developmental contexts. Here we investigate the set of genes associated with the development of eyespot color patterns, a trait that originated once within the Nymphalid family of butterflies. Although several genes associated with eyespot development have been identified, the eyespot gene regulatory network remains largely unknown.<h4>Results</h4>In this study, next-generation sequencing and transcriptome analyses were used to identify a large set of genes associated with eyespot development of Bicyclus anynana butterflies, at 3-6 h after pupation, prior to the differentiation of the color rings. Eyespot-associated genes were identified by comparing the transcriptomes of homologous micro-dissected wing tissues that either develop or do not develop eyespots in wild-type and a mutant line of butterflies, Spotty, with extra eyespots. Overall, 186 genes were significantly up and down-regulated in wing tissues that develop eyespots compared to wing tissues that do not. Many of the differentially expressed genes have yet to be annotated. New signaling pathways, including the Toll, Fibroblast Growth Factor (FGF), extracellular signal-regulated kinase (ERK) and/or Jun N-terminal kinase (JNK) signaling pathways are associated for the first time with eyespot development. In addition, several genes involved in wound healing and calcium signaling were also found to be associated with eyespots.<h4>Conclusions</h4>Overall, this study provides the identity of many new genes and signaling pathways associated with eyespots, and suggests that the ancient wound healing gene regulatory network may have been co-opted to cells at the center of the pattern to aid in eyespot origins. New transcription factors that may be providing different identities to distinct wing sectors, and genes with sexually dimorphic expression in the eyespots were also identified.

The origin and diversification of evolutionary novelties-lineage-specific traits of new adaptive value-is one of the key issues in evolutionary developmental biology. However, comparative analysis of the genetic and developmental bases of such traits can be difficult when they have no obvious homologue in model organisms. The finding that the evolution of morphological novelties often involves the recruitment of pre-existing genes and/or gene networks offers the potential to overcome this challenge. Knowledge about shared developmental processes obtained from extensive studies in model organisms can then be used to understand the origin and diversification of lineage-specific structures. Here, we illustrate this approach in relation to eyespots on the wings of Bicyclus anynana butterflies. A number of spontaneous mutations isolated in the laboratory affect eyespots, lepidopteran-specific features, and also processes that are shared by most insects. We discuss how eyespot mutants with disturbed embryonic development may help elucidate the genetic pathways involved in eyespot formation, and how venation mutants with altered eyespot patterns might shed light on mechanisms of eyespot development.

Abstract Butterfly eyespots are beautiful novel traits with an unknown developmental origin. Here we show that eyespots likely originated via co-option of the antennal gene-regulatory network (GRN) to novel locations on the wing. Using comparative transcriptome analysis, we show that eyespots cluster with antennae relative to multiple other tissues. Furthermore, three genes essential for eyespot development ( Distal-less ( Dll ), spalt ( sal ), and Antennapedia ( Antp )) share similar regulatory connections as those observed in the antennal GRN. CRISPR knockout of cis -regulatory elements (CREs) for Dll and sal led to the loss of eyespots and antennae, and also legs and wings, demonstrating that these CREs are highly pleiotropic. We conclude that eyespots likely re-used the ancient antennal GRN, a network previously implicated also in the development of legs and wings.

In butterflies there is a class of "intervein" wing patterns that have lines of symmetry halfway between wing veins. These patterns occur in a range of shapes, including eyespots, ellipses, and midlines, and were proposed to have evolved through developmental shifts along a midline-to-eyespot continuum. Here we show that Notch (N) upregulation, followed by activation of the transcription factor Distal-less (Dll), is an early event in the development of eyespot and intervein midline patterns across multiple species of butterflies. A relationship between eyespot phenotype and N and Dll expression is demonstrated in a loss-of-eyespot mutant in which N and Dll expression is reduced at missing eyespot sites. A phylogenetic comparison of expression time series from eight moth and butterfly species suggests that intervein N and Dll patterns are a derived characteristic of the butterfly lineage. Furthermore, prior to eyespot determination in eyespot-bearing butterflies, N and Dll are transiently expressed in a pattern that resembles ancestral intervein midline patterns. In this study we establish N upregulation as the earliest known event in eyespot determination, demonstrate gene expression associated with intervein midline color patterns, and provide molecular evidence that wing patterns evolved through addition to and truncation of a conserved midline-to-eyespot pattern formation sequence.