In the ME method, distance measures that correct for multiple hits at the same sites are used, and a topology showing the smallest value of the sum of all branches (S) is chosen as an estimate of the correct tree. However, the construction of a minimum evolution tree is time-consuming because, in principle, the S values for all topologies must be evaluated. The number of possible topologies (unrooted trees) rapidly increases with the number of taxa so it becomes very difficult to examine all topologies. In this case, one may use the neighbor-joining method. While the NJ tree is usually the same as the ME tree, when the number of taxa is small the difference between the NJ and ME trees can be substantial (reviewed in Nei and Kumar 2000). In this case if a long DNA or amino acid sequence is used, the ME tree is preferable. When the number of nucleotides or amino acids used is relatively small, the NJ method generates the correct topology more often than does the ME method (Nei et al. 1998, Takahashi and Nei 2000). In MEGA, we have provided the close-neighbor-interchange search to examine the neighborhood of the NJ tree to find the potential ME tree.