Traits that appear discontinuously along phylogenies may be explained by independent origins (homoplasy) or repeated loss (homology). While discriminating between these models is difficult, the dissection of gene regulatory networks (GRNs) which drive the development of such repeatedly occurring traits can offer a mechanistic window on this fundamental problem. The GRN responsible for the male-specific pattern of Drosophila (D.) melanogaster melanic tergite pigmentation has received considerable attention. In this system, a metabolic pathway of pigmentation enzyme genes is expressed in spatial and sex-specific (i.e., dimorphic) patterns. The dimorphic expression of several genes is regulated by the Bab transcription factors, which suppress pigmentation enzyme expression in females, by virtue of their high expression in this sex. Here, we analyzed the phylogenetic distribution of species with male-specific pigmentation and show that this dimorphism is phylogenetically widespread among fruit flies. The analysis of pigmentation enzyme gene expression in distantly related dimorphic and monomorphic species shows that dimorphism is driven by the similar deployment of a conserved metabolic pathway. However, sexually dimorphic Bab expression was found only in D. melanogaster and its close relatives. These results suggest that dimorphism evolved by parallel deployment of differentiation genes, but was derived through distinct architectures at the level of regulatory genes. This work demonstrates the interplay of constraint and flexibility within evolving GRNs, findings that may foretell the mechanisms of homoplasy more broadly.

A challenge for evolutionary research is to uncover how new morphological traits evolve the coordinated spatial and temporal expression patterns of genes that govern their formation during development. Detailed studies are often limited to characterizing how one or a few genes contributed to a trait's emergence, and thus our knowledge of how entire GRNs evolve their coordinated expression of each gene remains unresolved. The melanic color patterns decorating the male abdominal tergites of Drosophila (D.) melanogaster evolved in part by novel expression patterns for genes acting at the terminus of a pigment metabolic pathway, driven by cis-regulatory elements (CREs) with distinct mechanisms of Hox regulation. Here, we examined the expression and evolutionary histories of two important enzymes in this pathway, encoded by the pale and Ddc genes. We found that while both genes exhibit dynamic patterns of expression, a robust pattern of Ddc expression specifically evolved in the lineage of fruit flies with pronounced melanic abdomens. Derived Ddc expression requires the activity of a CRE previously shown to activate expression in response to epidermal wounding. We show that a binding site for the Grainy head transcription factor that promotes the ancestral wound healing function of this CRE is also required for abdominal activity. Together with previous findings in this system, our work shows how the GRN for a novel trait emerged by assembling unique yet similarly functioning CREs from heterogeneous starting points.

Changes in gene expression are a prominent feature of morphological evolution. These changes occur to hierarchical gene regulatory networks (GRNs) of transcription factor genes that regulate the expression of trait‐building differentiation genes. While changes in the expression of differentiation genes are essential to phenotypic evolution, they can be caused by mutations within cis‐regulatory elements (CREs) that drive their expression (cis‐evolution) or within genes for CRE‐interacting transcription factors (trans‐evolution). Locating these mutations remains a challenge, especially when experiments are limited to one species that possesses the ancestral or derived phenotype. We investigated CREs that control the expression of the differentiation genes tan and yellow, the expression of which evolved during the gain, modification, and loss of dimorphic pigmentation among Sophophora fruit flies. We show these CREs to be necessary components of a pigmentation GRN, as deletion from Drosophila melanogaster (derived dimorphic phenotype) resulted in lost expression and lost male‐specific pigmentation. We evaluated the ability of orthologous CRE sequences to drive reporter gene expression in species with modified (Drosophila auraria), secondarily lost (Drosophila ananassae), and ancestrally absent (Drosophila willistoni) pigmentation. We show that the transgene host frequently determines CRE activity, implicating trans‐evolution as a significant factor for this trait's diversity. We validated the gain of dimorphic Bab transcription factor expression as a trans‐change contributing to the dimorphic trait. Our findings suggest an amenability to change for the landscape of trans‐regulators and begs for an explanation as to why this is so common compared to the evolution of differentiation gene CREs.

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