previously gone unnoticed. In the 1980s early molecular work in the field was conducted by Norman R. Pace and colleagues, who used PCR to explore the diversity of ribosomal RNA sequences. The insights gained from these breakthrough studies led Pace to propose the idea of cloning DNA directly from environmental samples as early as 1985. This led to the first report of isolating and cloning bulk DNA from an environmental sample, published by Pace and colleagues in 1991 while Pace was in the Department of Biology at Indiana University. Considerable efforts ensured that these were not PCR false positives and supported the existence of a complex community of unexplored species. Although this methodology was limited to exploring highly conserved, non-protein coding genes, it did support early microbial morphology-based observations that diversity was far more complex than was known by culturing methods. Soon after that in 1995, Healy reported the metagenomic isolation of functional genes from "zoolibraries" constructed from a complex culture of environmental organisms grown in the laboratory on dried grasses. After leaving the Pace laboratory, Edward DeLong continued in the field and has published work that has largely laid the groundwork for environmental phylogenies based on signature 16S sequences, beginning with his group's construction of libraries from marine samples. In 2002, Mya Breitbart, Forest Rohwer, and colleagues used environmental shotgun sequencing (see below) to show that 200 liters of seawater contains over 5000 different viruses. Subsequent studies showed that there are more than a thousand viral species in human stool and possibly a million different viruses per kilogram of marine sediment, including many bacteriophages. Essentially all of the viruses in these studies were new species. In 2004, Gene Tyson, Jill Banfield, and colleagues at the University of California, Berkeley and the Joint Genome Institute sequenced DNA extracted from an acid mine drainage system. This effort resulted in the complete, or nearly complete, genomes for a handful of bacteria and archaea that had previously resisted attempts to culture them. Beginning in 2003, Craig Venter, leader of the privately funded parallel of the Human Genome Project, has led the Global Ocean Sampling Expedition (GOS), circumnavigating the globe and collecting metagenomic samples throughout the journey. All of these samples were sequenced using shotgun sequencing, in hopes that new genomes (and therefore new organisms) would be identified. The pilot project, conducted in the Sargasso Sea, found DNA from nearly 2000 different species, including