Tufte explains that "visually stratifying" the data within the image is a very powerful way to convey the data. Layering and separation allows the eyes of the audience to decipher between two or more pieces of critical information without confusion. Imagine if everything in a graph or diagram was all the same color. Your eyes would not know where to be drawn to first. Tufte says, " What matters-inevitably, unrelentingly-is the proper relationship among layers. These visual relationships must be in relevant proportion and in harmony to the substance of the ideas, evidence, and data conveyed." The point that Tufte is trying to make is that all of these layers have a relationship with one another, which allows the audience to make the connections between them in order to understand the meaning of the visual or graph.
See the link below for a great example of layering and separation in the design of this map of a mall.