Amoebozoa is the eukaryotic supergroup sister to Obazoa, the lineage that contains the animals and Fungi, as well as their protistan relatives, and the breviate and apusomonad flagellates. Amoebozoa is extraordinarily diverse, encompassing important model organisms and significant pathogens. Although amoebozoans are integral to global nutrient cycles and present in nearly all environments, they remain vastly understudied. We present a robust phylogeny of Amoebozoa based on broad representative set of taxa in a phylogenomic framework (325 genes). By sampling 61 taxa using culture-based and single-cell transcriptomics, our analyses show two major clades of Amoebozoa, Discosea, and Tevosa. This phylogeny refutes previous studies in major respects. Our results support the hypothesis that the last common ancestor of Amoebozoa was sexual and flagellated, it also may have had the ability to disperse propagules from a sporocarp-type fruiting body. Overall, the main macroevolutionary patterns in Amoebozoa appear to result from the parallel losses of homologous characters of a multiphase life cycle that included flagella, sex, and sporocarps rather than independent acquisition of convergent features.

Highlights d Arcellinida testate amoebae are comprised of seven major lineages d Reconstructed hypothetical ancestral states are congruent with Tonian fossils d Combined analysis of phylogenies and fossils suggest divergence as early as 730 mya

Phylogenomic analyses of hundreds of protein-coding genes aimed at resolving phylogenetic relationships is now a common practice. However, no software currently exists that includes tools for dataset construction and subsequent analysis with diverse validation strategies to assess robustness. Furthermore, there are no publicly available high-quality curated databases designed to assess deep (>100 million years) relationships in the tree of eukaryotes. To address these issues, we developed an easy-to-use software package, PhyloFisher (https://github.com/TheBrownLab/PhyloFisher), written in Python 3. PhyloFisher includes a manually curated database of 240 protein-coding genes from 304 eukaryotic taxa covering known eukaryotic diversity, a novel tool for ortholog selection, and utilities that will perform diverse analyses required by state-of-the-art phylogenomic investigations. Through phylogenetic reconstructions of the tree of eukaryotes and of the Saccharomycetaceae clade of budding yeasts, we demonstrate the utility of the PhyloFisher workflow and the provided starting database to address phylogenetic questions across a large range of evolutionary time points for diverse groups of organisms. We also demonstrate that undetected paralogy can remain in phylogenomic “single-copy orthogroup” datasets constructed using widely accepted methods such as all vs. all BLAST searches followed by Markov Cluster Algorithm (MCL) clustering and application of automated tree pruning algorithms. Finally, we show how the PhyloFisher workflow helps detect inadvertent paralog inclusions, allowing the user to make more informed decisions regarding orthology assignments, leading to a more accurate final dataset.

29Recent phylogenetic analyses position certain 'orphan' protist lineages deep in the tree of 30 eukaryotic life, but their exact placements are poorly resolved. We conducted phylogenomic 31 analyses that incorporate deeply sequenced transcriptomes from representatives of 32 collodictyonids (diphylleids), rigifilids, Mantamonas and ancyromonads (planomonads). 33Analyses of 351 genes, using site-heterogeneous mixture models, strongly support a novel 34 supergroup-level clade that includes collodictyonids, rigifilids and Mantamonas, which we name 35 'CRuMs'. Further, they robustly place CRuMs as the closest branch to Amorphea (including 36 animals and fungi). Ancyromonads are strongly inferred to be more distantly related to 37 Amorphea than are CRuMs. They emerge either as sister to malawimonads, or as a separate 38 deeper branch. CRuMs and ancyromonads represent two distinct major groups that branch 39 deeply on the lineage that includes animals, near the most commonly inferred root of the 40 eukaryote tree. This makes both groups crucial in examinations of the deepest-level history of 41 extant eukaryotes. 42 43 Introduction 44