The Holozoa clade comprises animals and several unicellular lineages (choanoflagellates, filastereans and teretosporeans). Understanding their full diversity is essential to address the origins of animals and other evolutionary questions. However, they are poorly known. To provide more insights into the real diversity of holozoans and check for undiscovered diversity, we here analysed 18S rDNA metabarcoding data from the global Tara Oceans expedition. To overcome the low phylogenetic information contained in the metabarcoding dataset (composed of sequences from the short V9 region of the gene), we used similarity networks by combining two datasets: unknown environmental sequences from Tara Oceans and known reference sequences from GenBank. We then calculated network metrics to compare environmental to reference sequences. These metrics reflected the divergence between both types of sequences and provided an effective way to search for evolutionary relevant diversity, further validated by phylogenetic placements. Our results showed that the percentage of unicellular holozoan diversity remains hidden. We found novelties in several lineages, especially in Acanthoecida choanoflagellates. We also identified a potential new holozoan group that could not be assigned to any of the described extant clades. Data on geographical distribution showed that, although ubiquitous, each unicellular holozoan lineage exhibits a different distribution pattern. We also identified a positive association between new animal hosts and the ichthyosporean symbiont Creolimax fragrantissima, as well as for other holozoans previously reported as free-living. Overall, our analyses provide a fresh perspective into the diversity and ecology of unicellular holozoans, highlighting the amount of undescribed diversity.

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