Our understanding of the origin of animals has been transformed by characterizing their most closely related, unicellular sisters: the choanoflagellates, filastereans, and ichthyosporeans. Together with animals, these lineages make up the Holozoa [1, 2]. Many traits previously considered "animal specific" were subsequently found in other holozoans [3, 4], showing that they evolved before animals, although exactly when is currently uncertain because several key relationships remain unresolved [2, 5]. Here we report the morphology and transcriptome sequencing from three novel unicellular holozoans: Pigoraptor vietnamica and Pigoraptor chileana, which are related to filastereans, and Syssomonas multiformis, which forms a new lineage with Corallochytrium in phylogenomic analyses. All three species are predatory flagellates that feed on large eukaryotic prey, and all three also appear to exhibit complex life histories with several distinct stages, including multicellular clusters. Examination of genes associated with multicellularity in animals showed that the new filastereans contain a cell-adhesion gene repertoire similar to those of other species in this group. Syssomonas multiformis possessed a smaller complement overall but does encode genes absent from the earlier-branching ichthyosporeans. Analysis of the T-box transcription factor domain showed expansion of T-box transcription factors based on combination with a non-T-box domain (a receiver domain), which has not been described outside of vertebrates. This domain and other domains we identified in all unicellular holozoans are part of the two-component signaling system that has been lost in animals, suggesting the continued use of this system in the closest relatives of animals and emphasizing the importance of studying loss of function as well as gain in major evolutionary transitions.

Biology Centre Institute of Parasitology Czech Academy of Sciences Branisovska 31 370 05 Ceske Budejovice Czech Republic

Department of Botany University of British Columbia 3529 6270 University Boulevard Vancouver BC V6T 1Z4 Canada

Department of Microbiology Epidemiology and Infectious Diseases Penza State University Lermontov Street 37 Penza 440026 Russia

Laboratory of Microbiology Institute for Biology of Inland Waters Russian Academy of Sciences Yaroslavl Region Borok 152742 Russia

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