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Pulsed-field electrophoresis (PFGE) of the SspI genomic digests of 14 Thennus isolates showed that each one had a unique restriction enzyme digestion pattern. A group of New Zealand strains showed some shared bands, but each isolate gave essentially a unique fingerprint. In addition, evolutionary distances between Thermus strains estimated by using PFGE restriction fragment length polymorphisms (PFGE-RFLPs) correlate well with those based on small-subunit rRNA sequence data. As a consequence, the phylogenetic trees constructed on the basis of PFGE-RFLPs and those constructed by using small-subunit rRNA sequences generally agree. On the basis of the evolutionary distances estimated by using PFGE-RFLPs, the estimated average genomic rate of divergence for Themus spp. is approximately 0.27% per million years. Many strains of thermophilic bacteria isolated from hot springs located around th'e world have been classified as belonging to the genus Thermus, but the relationships among these strains have not been well established. Only three species of Thermus have been validly described so far (3, 11, 15): Thermus aquaticus, originally isolated from hot springs in Yellowstone National Park; Thermus Jiliformis, a single strain isolated in New Zealand; and Thermus ruber, isolated from many locations, and distinguished from other Thermus strains by red pigmentation and a lower growth temperature. "Ther- mus thermophilus" (20) is thought to be a separate species, though it has not been formally accepted. Several Thermus isolates show high DNA-DNA homology with HB8, the "T. thermophilus" type strain (28). Whether all the Thermus iso- lates obtained are taxonomic variants of the three nominal species or are species themselves is the source of some debate. A number of studies have attempted to resolve the status of Thermus strains. Phenetic studies have been performed to classify Themus isolates from New Zealand (lo), Iceland (4), and Portugal (22) on the basis of microbiological and pheno- typic characters. In addition, a phenetic classification of 131 Thermus strains isolated from hot springs around the world has been performed (12). On the basis of analysis of 68 phenotypic characteristics, eight distinct groups of Thermus strains were defined at the level of similarity separating the three recog- nized species. Classifications based on numerical taxonomy, however, often fail to identify groups that share close evolu- tionary relationships, and it is precisely these relationships that many systematists feel should form the basis of any classifica- tion. Therefore, another approach to resolving the problems with the classification of the Thermus strains is to use procedures explicitly designed to uncover the evolutionary relationships or phylogeny of the taxa in question. In a recent paper, Saul et al. (23) used small-subunit (SSU) rRNA sequences of 20 Thermus strains to reconstruct their phylogenetic history. The study showed that of the 20 strains, there were four primary clades

Pulsed-Field Gel Electrophoresis of Genomic Digests of Thermus Strains and Its Implications for Taxonomic and Evolutionary Studies