Mutations to gene regulatory networks drive evolutionary adaptation, but mutations can also occur without phenotypic change. These neutral mutations instead lead to developmental systems drift, evolutionary divergence in developmental systems that does not alter the traits produced. Here we examine developmental systems drift between two tunicate species, Corella inflata and Ciona robusta, in the cardiopharyngeal gene regulatory network. Through cross-species assays and functional enhancer analysis, we assess the amount of drift both in cis and in trans. Here we show that the trans-regulatory architecture of the cardiopharyngeal gene regulatory network is largely conserved between C. robusta and C. inflata, but cis-regulatory elements within this network exhibit distinct levels of conservation. These results suggest that the amount of drift cis-regulatory elements undergo is not governed by overarching principles but rather by distinct structural and functional constraints which are unique to each cis-regulatory element. We show that the enhancer for FoxF, a key cardiopharyngeal gene, is highly conserved and propose a model for the unique structural and functional constraints which this cis-regulatory element experiences
