Description: The phylum Cercozoa consists of a diverse assemblage of amoeboid and flagellated protists that forms a major component of the supergroup, Rhizaria. However, despite its size and ubiquity, the phylogeny of the Cercozoa remains unclear as morphological variability between cercozoan species and ambiguity in molecular analyses, including phylogenomic approaches, have produced ambiguous results and raised doubts about the monophyly of the group. Here we sought to resolve these ambiguities using a 161-gene phylogenetic dataset with data from newly available genomes and deeply sequenced transcriptomes, including three new transcriptomes from Aurigamonas solis, Abollifer prolabens, and a novel species, Lapot gusevi n. gen. n. sp. Our phylogenomic analysis strongly supported a monophyletic Cercozoa, and approximately-unbiased tests rejected the paraphyletic topologies observed in previous studies. The transcriptome of L. gusevi represents the first transcriptomic data from the large and recently characterized Aquavolonidae-Treumulida-'Novel Clade 12' group, and phylogenomics supported its position as sister to the cercozoan subphylum, Endomyxa. These results provide insights into the phylogeny of the Cercozoa and the Rhizaria as a whole.

Description: Background: The origin of animals from their unicellular ancestor was one of the most important events in evolutionary history, but the nature and the order of events leading up to the emergence of multicellular animals are still highly uncertain. The diversity and biology of unicellular relatives of animals have strongly informed our understanding of the transition from single-celled organisms to the multicellular Metazoa. Here, we analyze the cellular structures and complex life cycles of the novel unicellular holozoans Pigoraptor and Syssomonas (Opisthokonta), and their implications for the origin of animals. Results: Syssomonas and Pigoraptor are characterized by complex life cycles with a variety of cell types including flagellates, amoeboflagellates, amoeboid non-flagellar cells, and spherical cysts. The life cycles also include the formation of multicellular aggregations and syncytium-like structures, and an unusual diet for single-celled opisthokonts (partial cell fusion and joint sucking of a large eukaryotic prey), all of which provide new insights into the origin of multicellularity in Metazoa. Several existing models explaining the origin of multicellular animals have been put forward, but these data are interestingly consistent with one, the "synzoospore hypothesis." Conclusions: The feeding modes of the ancestral metazoan may have been more complex than previously thought, including not only bacterial prey, but also larger eukaryotic cells and organic structures. The ability to feed on large eukaryotic prey could have been a powerful trigger in the formation and development of both aggregative (e.g., joint feeding, which also implies signaling) and clonal (e.g., hypertrophic growth followed by palintomy) multicellular stages that played important roles in the emergence of multicellular animals.

Description: Highlights: • New predatory protist (Tunicaraptor) with unique morphology is related to animals • Tunicaraptor calls into question many of the well-accepted relationships in Holozoa • Tunicaraptor possesses a unique combination of “animal-specific” gene products • Eukaryovorous flagellates may represent a major share of unicellular animal relatives The origin of animals is one of the most intensely studied evolutionary events, and our understanding of this transition was greatly advanced by analyses of unicellular relatives of animals, which have shown many “animal-specific” genes actually arose in protistan ancestors long before the emergence of animals [1–3]. These genes have complex distributions, and the protists have diverse lifestyles, so understanding their evolutionary significance requires both a robust phylogeny of animal relatives and a detailed understanding of their biology [4, 5]. But discoveries of new animal-related lineages are rare and historically biased to bacteriovores and parasites. Here, we characterize the morphology and transcriptome content of a new animal-related lineage, predatory flagellate Tunicaraptor unikontum. Tunicaraptor is an extremely small (3–5 μm) and morphologically simple cell superficially resembling some fungal zoospores, but it survives by preying on other eukaryotes, possibly using a dedicated but transient “mouth,” which is unique for unicellular opisthokonts. The Tunicaraptor transcriptome encodes a full complement of flagellar genes and the flagella-associated calcium channel, which is only common to predatory animal relatives and missing in microbial parasites and grazers. Tunicaraptor also encodes several major classes of animal cell adhesion molecules, as well as transcription factors and homologs of proteins involved in neurodevelopment that have not been found in other animal-related lineages. Phylogenomics, including Tunicaraptor, challenges the existing framework used to reconstruct the evolution of animal-specific genes and emphasizes that the diversity of animal-related lineages may be better understood only once the smaller, more inconspicuous animal-related lineages are better studied.

Description: Rhodophyta (red algae) is one of three lineages of Archaeplastida1, a supergroup that is united by the primary endosymbiotic origin of plastids in eukaryotes2,3. Red algae are a diverse and species-rich group, members of which are typically photoautotrophic, but are united by a number of highly derived characteristics: they have relatively small intron-poor genomes, reduced metabolism and lack cytoskeletal structures that are associated with motility, flagella and centrioles. This suggests that marked gene loss occurred around their origin4; however, this is difficult to reconstruct because they differ so much from the other archaeplastid lineages, and the relationships between these lineages are unclear. Here we describe the novel eukaryotic phylum Rhodelphidia and, using phylogenomics, demonstrate that it is a closely related sister to red algae. However, the characteristics of the two Rhodelphis species described here are nearly opposite to those that define red algae: they are non-photosynthetic, flagellate predators with gene-rich genomes, along with a relic genome-lacking primary plastid that probably participates in haem synthesis. Overall, these findings alter our views of the origins of Rhodophyta, and Archaeplastida evolution as a whole, as they indicate that mixotrophic feeding—that is, a combination of predation and phototrophy—persisted well into the evolution of the group.

Description: 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.

Description: Highlights: • Generation of transcriptomes from three new Pirsoniales species for the first time. • Generation of transcriptomes from four new Developea species, enriching the clade. • Phylogenomic recovery of a monophyletic Bigyromonadea: Pirsoniales and Developea. • Bigyormonads’ ability to aggregate and form pseudopod resembles oomycete zoospores. • Report of the first case of eukaryovory in the flagellated stages of Pirsoniales. Stramenopiles are a diverse but relatively well-studied eukaryotic supergroup with considerable genomic information available (Sibbald and Archibald, 2017). Nevertheless, the relationships between major stramenopile subgroups remain unresolved, in part due to a lack of data from small nanoflagellates that make up a lot of the genetic diversity of the group. This is most obvious in Bigyromonadea, which is one of four major stramenopile subgroups but represented by a single transcriptome. To examine the diversity of Bigyromonadea and how the lack of data affects the tree, we generated transcriptomes from seven novel bigyromonada species described in this study: Develocauda condao n. gen. n. sp., Develocanicus komovi n. gen. n. sp., Develocanicus vyazemskyi n. sp., Cubaremonas variflagellatum n. gen. n. sp., Pirsonia chemainus nom. prov., Feodosia pseudopoda nom. prov., and Koktebelia satura nom. prov. Both maximum likelihood and Bayesian phylogenomic trees based on a 247 genematrix recovered a monophyletic Bigyromonadea that includes two diverse subgroups, Developea and Pirsoniales, that were not previously related based on single gene trees. Maximum likelihood analyses show Bigyromonadea related to oomycetes, whereas Bayesian analyses and topology testing were inconclusive. We observed similarities between the novel bigyromonad species and motile zoospores of oomycetes in morphology and the ability to self-aggregate. Rare formation of pseudopods and fused cells were also observed, traits that are also found in members of labyrinthulomycetes, another osmotrophic stramenopiles. Furthermore, we report the first case of eukaryovory in the flagellated stages of Pirsoniales. These analyses reveal new diversity of Bigyromonadea, and altogether suggest their monophyly with oomycetes, collectively known as Pseudofungi, is the most likely topology of the stramenopile tree.