Philip Hugenholtz reviews the challenges and progress in understanding prokaryotic diversity in the genomic era. The study highlights a significant sampling bias towards four bacterial phyla—Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes—due to the dominance of culturable microorganisms in research. Cultivated microorganisms are often not numerically dominant in their environments, leading to an underrepresentation of prokaryotic diversity. The "great plate-count anomaly" suggests that less than 1% of microbial species are culturable, raising questions about the representativeness of cultivated microorganisms. The use of molecular phylogenetic methods, such as 16S rRNA analysis, has helped overcome this bias by directly accessing microbial genomes from environmental samples. This approach has revealed a much greater diversity of prokaryotic phyla than previously thought, with estimates ranging from 35 to 45. The analysis of 16S rRNA sequences has also led to the identification of new lineages, including candidate phyla with environmental clone sequences. The study discusses the challenges of genome sequencing for uncultivated prokaryotes and the need for a more comprehensive sampling of phyla to understand prokaryotic diversity. It also explores the potential of phylogenetically directed isolation strategies to cultivate novel environmental lineages. Techniques such as FISH and PCR are used to monitor and isolate target organisms. The discovery of proteorhodopsin in an uncultivated marine bacterioplankton lineage highlights the importance of environmental genomic approaches. The review emphasizes the need for further research to understand the evolutionary history and physiological characteristics of uncultivated prokaryotes. It concludes that many prokaryotic phyla remain uncharacterized and that direct access to their genomes is essential for a complete understanding of microbial diversity. The study underscores the importance of integrating molecular and cultivation-based approaches to overcome the limitations of traditional microbiological methods.Philip Hugenholtz reviews the challenges and progress in understanding prokaryotic diversity in the genomic era. The study highlights a significant sampling bias towards four bacterial phyla—Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes—due to the dominance of culturable microorganisms in research. Cultivated microorganisms are often not numerically dominant in their environments, leading to an underrepresentation of prokaryotic diversity. The "great plate-count anomaly" suggests that less than 1% of microbial species are culturable, raising questions about the representativeness of cultivated microorganisms. The use of molecular phylogenetic methods, such as 16S rRNA analysis, has helped overcome this bias by directly accessing microbial genomes from environmental samples. This approach has revealed a much greater diversity of prokaryotic phyla than previously thought, with estimates ranging from 35 to 45. The analysis of 16S rRNA sequences has also led to the identification of new lineages, including candidate phyla with environmental clone sequences. The study discusses the challenges of genome sequencing for uncultivated prokaryotes and the need for a more comprehensive sampling of phyla to understand prokaryotic diversity. It also explores the potential of phylogenetically directed isolation strategies to cultivate novel environmental lineages. Techniques such as FISH and PCR are used to monitor and isolate target organisms. The discovery of proteorhodopsin in an uncultivated marine bacterioplankton lineage highlights the importance of environmental genomic approaches. The review emphasizes the need for further research to understand the evolutionary history and physiological characteristics of uncultivated prokaryotes. It concludes that many prokaryotic phyla remain uncharacterized and that direct access to their genomes is essential for a complete understanding of microbial diversity. The study underscores the importance of integrating molecular and cultivation-based approaches to overcome the limitations of traditional microbiological methods.