Outgroup Rooting of an unrooted network

    Given an unrooted network of relationships [based on the 1,140 bp DNA sequence of mtDNA Cytochrome b gene] among four species of Carnivora in the suborder Caniformia, outgroup rooting uses an additional taxon (an outgroup) known from independent evidence to be less closely related to any of the other species (the ingroup) than they are to each other. The analysis is repeated with the five taxa, and the placement of the outgroup among the ingroup determines the root.

    In the example, Felis is a carnivore in a different suborder (Feliformia) than the other four species (Caniformia). Inclusion of Felis in the network analysis places the root on the internode, as expected if Ursus and Ailuropoda are each others closest relatives in the bear family, Ursidae.

    This method requires accurate information as to ingroup / outgroup relationships. Ideally, the outgroup should be the sister taxon, which is the most closely related taxon not included in the group. If a more distant outgroup taxon is used, the accumulated differences from the ingroup may obscure the actual similarities. Note that in this example, the bootstrap support for the branch leading to the two Ursidae has fallen to 90% from 99% in the unrooted network.