loading . . . Global Metagenomics Reveals Hidden Protist Diversity Protists, defined as unicellular eukaryotes distinct from animals, plants, and fungi, are a polyphyletic group that predominates the eukaryotic tree of life, exhibiting significant phylogenetic diversity and fulfilling critical ecological roles. Historically, research has prioritized protists associated with animals and plants, particularly those of medical significance, thereby overlooking the majority of protist diversity. Conventional molecular methods, such as 18S rRNA gene amplicon sequencing, frequently encounter limitations, including primer binding bias and PCR bias caused by gene length variations, creating a biased perspective of understudied protistan diversity. Further, most protist lineages are notoriously difficult to cultivate. Here, we apply a cultivation-independent approach in which we analyzed over 27,000 assembled metagenomes and protist single cell genomes and 21 long 18S rRNA gene amplicon data sets from various global ecosystems, including marine, freshwater, and soil environments. We recovered 157,958 18S rRNA gene sequences (≥800 bp), which clustered into 103,338 operational taxonomic units (OTUs) at 97% sequence identity and 24,438 OTUs at 85% identity. Notably, 69% of 13,238 non-singleton clusters at 85% identity consisted exclusively of environmental sequences, uncovering a wealth of yet uncultivated and unclassified novel protist diversity. A comprehensive taxonomic framework of eukaryotes based on concatenated 18S and 28S rRNA genes that incorporated most novel lineages revealed substantial underrepresentation of Amoebozoa, Discoba, and Rhizaria in reference databases, with many lacking isolate or genome sequence representation. Further, we identified 13 eukaryotic lineages with novelty on higher taxonomic ranks, such as class and phylum-level, that lack representation in public databases. The corresponding 85% OTUs were primarily affiliated with Excavata, with some branching closely to the root of the eukaryotic tree. Comprehensive analysis of the global distribution of eukaryotes revealed uneven microbial eukaryotic diversity across supergroups and ecosystems, with notable novelty particularly in soil and marine environments. We then examined co-occurrence between protists and prokaryotes, predicting putative symbiotic or predator-prey relationships, particularly among understudied protist groups with bacteria such as Verrucomicrobia and Rickettsiales. Our results substantially enhance the understanding of protistan diversity and distribution, revealing taxonomic blind spots and laying groundwork for future studies of these organisms' ecological roles. ### Competing Interest Statement The authors have declared no competing interest. https://www.biorxiv.org/content/10.1101/2025.03.26.645542v1
