Animals and fungi have radically distinct morphologies, yet both evolved within the same eukaryotic supergroup: Opisthokonta1,2. Here we reconstructed the trajectory of genetic changes that accompanied the origin of Metazoa and Fungi since the divergence of Opisthokonta with a dataset that includes four novel genomes from crucial positions in the Opisthokonta phylogeny. We show that animals arose only after the accumulation of genes functionally important for their multicellularity, a tendency that began in the pre-metazoan ancestors and later accelerated in the metazoan root. By contrast, the pre-fungal ancestors experienced net losses of most functional categories, including those gained in the path to Metazoa. On a broad-scale functional level, fungal genomes contain a higher proportion of metabolic genes and diverged less from the last common ancestor of Opisthokonta than did the gene repertoires of Metazoa. Metazoa and Fungi also show differences regarding gene gain mechanisms. Gene fusions are more prevalent in Metazoa, whereas a larger fraction of gene gains were detected as horizontal gene transfers in Fungi and protists, in agreement with the long-standing idea that transfers would be less relevant in Metazoa due to germline isolation3–5. Together, our results indicate that animals and fungi evolved under two contrasting trajectories of genetic change that predated the origin of both groups. The gradual establishment of two clearly differentiated genomic contexts thus set the stage for the emergence of Metazoa and Fungi.

Opisthokonta represents a major lineage of eukaryotes and includes fungi and metazoans, as well as other less known unicellular groups. The latter are paraphyletic assemblages that branch in between the former two groups, and thus are important for understanding the origin and early diversification of opisthokonts. The full range of their diversity, however, has not yet been explored from diverse ecological habitats. Freshwater environments are crucial sources for new diversity; they are considered even more heterogeneous than marine ecosystems. This heterogeneity implies more ecological niches where local eukaryotic communities are located. However, knowledge of the unicellular opisthokont diversity is scarce from freshwater environments. Here, we performed an 18S rDNA metabarcoding study in the Middle Paraná River, Argentina, to characterize the molecular diversity of microbial eukaryotes, in particular unicellular members of Opisthokonta. We identified a potential novel clade branching as a sister-group to Fungi. We also detected in our data that more than 60% operational taxonomic units classified as unicellular holozoans (animals and relatives) represent new taxa at the species level. Of the remaining, the majority was assigned to the newly described holozoan species, Syssomonas multiformis. Together, our results show that a large hidden diversity of unicellular members of opisthokonts still remain to be uncovered. We also found that the geographical and ecological distribution of several taxa considered exclusive to marine environments is wider than previously thought.

Animals with bilateral symmetry comprise the majority of the described species within Metazoa. However, the nature of the first bilaterian animal remains unknown. As most recent molecular phylogenies point to Xenacoelomorpha as the sister group to the rest of Bilateria, understanding their biology, ecology and diversity is key to reconstructing the nature of the last common bilaterian ancestor (Urbilateria). To date, sampling efforts have focused mainly on coastal areas, leaving potential gaps in our understanding of the full diversity of xenacoelomorphs. We therefore analysed 18S rDNA metabarcoding data from three marine projects covering benthic and pelagic habitats worldwide. Our results show that acoels have a greater richness in planktonic environments than previously described. Interestingly, we also identified a putative novel clade of acoels in the deep benthos that branches as sister group to the rest of Acoela, thus representing the earliest-branching acoel clade. Our data highlight deep-sea environments as an ideal habitat to sample acoels with key phylogenetic positions, which might be useful for reconstructing the early evolution of Bilateria.