Abstract:Many insects use vision to inform their behavior, but visual information differs between habitats and the sensory demands vary with each species' ecology. The small size of insects' eyes constrains their optical performance, and so it is unsurprising that they have evolved specializations for optimizing the information they obtain from their habitat. Unraveling how behavioral, environmental, and phylogenetic factors influence the evolution of such specializations is difficult, however, because existing techniques to analyze insect eyes require specimens to be preserved beforehand. To facilitate broad comparative studies on insect eyes and the evolution of complex visual behavior, we developed a novel analysis technique that uses x-ray micro-computed tomography to quantify and recreate the visual world of insects. We use our methodology to investigate the eyes of fungus gnats (Orfeliini), a tribe of diminutive Dipterans, to identify the visual specializations they evolved for surviving in different forest habitats and to explore how this changed over 30 million years of evolutionary history. The specimens we studied were preserved in different ways (in ethanol, air dried, and as an endocast in amber), demonstrating that our method provides a new opportunity to quantitatively study and compare the vision of a wide range insects held in museum collections. Our analysis indicates that different visual specializations have evolved between fungus gnat species living in different forest types and that the eyes of gnats from a similar geographic location have evolved to match the changing environmental conditions. Despite the small size of fungus gnats, evolution has evidentially been able to exploit sensory specializations to meet the differing sensory demands of species from a variety of forest habitats.Significance statement: Do insects have visual specializations that evolve with changes in their environment? To answer this question, a novel analysis technique is described that uses 3D imaging and simulations to compare the vision of ancient amber-embedded insects to those of their extant relatives. This study investigated the vision of fungus gnats to understand how tiny insects use vision to negotiate forests, some of the world's most visually complex environments. Despite being amongst the smallest of any flying insect, the gnats' miniature eyes have evolved visual specializations specifically adapted for different forest types, allowing different species to meet their visual demands of their specific habitats.Introduction: Vision provides essential information to meet the sensory demands of flying insects and is utilized to orchestrate both movement through, and interactions with, their environment (1). To fly safely and efficiently and to avoid collisions with obstacles, an insect must control its speed and orientation. To forage successfully, it must locate and recognize food objects from the wing, before safely approaching and landing upon them. The visual requirements of both sets of tasks depend up...