The benefits of increasing the number of microsatellites utilized in genetic population studies: an empirical perspective.

One of the key issues concerning the application of microsatellite DNA data in evolutionary studies is how the number of loci applied may influence the stability of genetic distances and corresponding phylograms. While computer simulations have suggested that over 30 microsatellites are required for accurate evolutionary inference, we show that a median of only six loci have been generally applied in studies of wild populations. Factors contributing to this contrast include: i) uncertainty regarding the potential benefits that can be gained from a realistic increase in the number of loci used; and ii) the lack of empirical studies assessing the influence of the number of microsatellites on the reliability of genetic distance estimation and phylogeny construction. In order to address these issues, we applied resampling techniques to microsatellite data in widely distributed populations of European grayling (Thymallus thymallus, Salmonidae). In agreement with expectations based on simulated data, we demonstrate empirically that the stability of commonly used genetic distances (DCE, DA and (deltamu)2) and the corresponding neighbor-joining phylograms is positively associated with the number of microsatellites utilized. For instance, increasing the number of loci from six to 17 resulted in a striking 75% increase in the proportion of DCE phylogram nodes supported by a bootstrap estimate of over 70%. Our results demonstrate that even moderately increasing the number of loci can be very beneficial--a finding extremely relevant for studies of natural populations for which optimally high microsatellite numbers are out of reach. Furthermore, the number of loci most commonly used to date may lead to erroneous inference of the evolutionary relationships between populations.