This is dependent on multiple site factors and the study question. In river systems for example, confluence points in rivers represent areas of water and DNA mixing of two potentially distinct fish communities. Therefore a point collected within the tributary as well as upstream and downstream of its confluence with the main stem may be required to determine fish community equivalence in the different parts of the river system. Similarly this logic could be applied to barriers or dams in rivers. Other site factors to consider would be pollution sources, major land use or habitat differences and differences in riparian vegetation.