Expanded iOn switch toolkit enables flexible clonal labeling and dynamic imaging in model and non-model animals.

Understanding how neural progenitors generate diverse neuronal subtypes is central to developmental and evolutionary neuroscience. Lineage tracing is a key tool for dissecting these processes, but existing methods are often constrained by stochastic expression, recombination bias, or reliance on transgenic models-limitations that restrict their application in non-model organisms. Here, we present an enhanced iOn switch system optimized for evo-devo research. Leveraging its modular, integration-based transgene expression logic, we developed a tunable labeling strategy that enables both sparse and dense labeling within a single framework. We further expanded the system's fluorescent palette and introduced subcellular targeting options to support multiclonal analysis and stable time-lapse imaging. Finally, we demonstrate the versatility of this approach across a wide range of vertebrate species, including mouse, guinea pig, rat, chick, turtle, and zebrafish, thereby establishing the iOn switch system as a flexible and accessible tool for evo-devo research.