The article by Philip Hugenholtz explores the challenges and advancements in understanding prokaryotic diversity, particularly in the genomic era. It highlights the significant sampling bias towards four bacterial phyla—Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes—out of the 35 bacterial and 18 archaeal phyla. This bias is often overlooked because microorganisms isolated in pure culture are typically those that grow rapidly and are easy to cultivate, leading to a focus on "microbial weeds" rather than the broader diversity of prokaryotes. Hugenholtz discusses the pioneering work of Carl Woese and colleagues in using small-subunit ribosomal RNA (16S rRNA) sequences to define evolutionary relationships and quantify diversity. This approach has led to the recognition of numerous new bacterial and archaeal lineages, challenging the traditional taxonomic classifications. The article also emphasizes the importance of environmental sampling and molecular techniques in accessing the genomes of uncultured microorganisms, which can provide insights into their physiological roles and evolutionary histories. The author outlines methods for cultivating novel environmental lineages, including the use of phylogenetically directed isolation strategies and physical isolation techniques. Additionally, he discusses the direct access to microbial genomes from environmental samples through cloning and sequencing, which has revealed novel functionalities, such as the discovery of proteorhodopsin in marine bacterioplankton. In conclusion, Hugenholtz argues that the study of uncharacterized prokaryotic phyla is crucial for understanding the full spectrum of microbial diversity and evolution. He calls for more efforts to sequence genomes from uncultured lineages, emphasizing the potential for these organisms to possess unique physiological repertoires and evolutionary histories.The article by Philip Hugenholtz explores the challenges and advancements in understanding prokaryotic diversity, particularly in the genomic era. It highlights the significant sampling bias towards four bacterial phyla—Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes—out of the 35 bacterial and 18 archaeal phyla. This bias is often overlooked because microorganisms isolated in pure culture are typically those that grow rapidly and are easy to cultivate, leading to a focus on "microbial weeds" rather than the broader diversity of prokaryotes. Hugenholtz discusses the pioneering work of Carl Woese and colleagues in using small-subunit ribosomal RNA (16S rRNA) sequences to define evolutionary relationships and quantify diversity. This approach has led to the recognition of numerous new bacterial and archaeal lineages, challenging the traditional taxonomic classifications. The article also emphasizes the importance of environmental sampling and molecular techniques in accessing the genomes of uncultured microorganisms, which can provide insights into their physiological roles and evolutionary histories. The author outlines methods for cultivating novel environmental lineages, including the use of phylogenetically directed isolation strategies and physical isolation techniques. Additionally, he discusses the direct access to microbial genomes from environmental samples through cloning and sequencing, which has revealed novel functionalities, such as the discovery of proteorhodopsin in marine bacterioplankton. In conclusion, Hugenholtz argues that the study of uncharacterized prokaryotic phyla is crucial for understanding the full spectrum of microbial diversity and evolution. He calls for more efforts to sequence genomes from uncultured lineages, emphasizing the potential for these organisms to possess unique physiological repertoires and evolutionary histories.