To combine a couple sentences on page 60, I would define layering as:
usually achieved by a hierarchy of visual effects, enhances representation of both data dimensionality and density on flatland
In Tufte's examples, layering is usually portrayed through the use of color to distinguish different aspects of the graphic such as with the industrial diagram..
Separation seems to involve the use of white space or lines. Separation can be used incorrectly or correctly. Such as, the use of white space to create vibration effects or the use of lines to create an overbearing grid.