DOI: 10.5061/dryad.vq83bk3q8
title: Phylogenomic analyses of 2,786 genes in 158 lineages support a root of the eukaryotic tree of life between opisthokonts and all other lineages

description: Advances in phylogenetic methods and high-throughput sequencing have
 allowed the reconstruction of deep phylogenetic relationships in the
 evolutionary history of eukaryotes. Yet, the root of the eukaryotic tree
 of life remains elusive. The most ‘popular’ (i.e. in textbooks and
 reviews) hypothesis for the root is between Unikonta (Opisthokonta +
 Amoebozoa) and Bikonta (all other eukaryotes), which emerged from analyses
 of a single gene fusion and a limited sampling of eukaryotic lineages.
 Subsequent highly-cited studies based on concatenation of genes supported
 this hypothesis with some variations or proposed a root within the
 Excavata. However, concatenation of genes neither considers
 phylogenetically-informative events (i.e. gene duplications and losses)
 nor provides an estimate of the root. A more recent study using gene
 tree-species tree reconciliation methods suggested the root lies between
 Opisthokonta and all other eukaryotes, but only including 59 taxa and 20
 genes. Here we apply a gene tree – species tree reconciliation approach to
 a gene-rich and taxon-rich dataset (i.e. 2,786 gene families from two sets
 of ~158 diverse eukaryotic lineages) to assess the root, and we iterate
 each analysis 100 times to quantify tree space uncertainty. Our results
 estimate a root between Fungi and all other eukaryotes, or between
 Opisthokonta and all other eukaryotes, and reject alternative popular
 roots from the literature. Based on further analysis of genome size, we
 propose Opisthokonta + others as the most likely root. Finding the root of
 the eukaryotic tree of life is critical for the field of comparative
 biology as it allows us to understand the timing and mode of evolution of
 characters across the evolutionary history of eukaryotes.
description: Here we provide the alignments, gene trees, inputs, and outputs
 from our project entitled "Phylogenomic Analyses of 2,786 Genes in
 158 Lineages Support a Root of The Eukaryotic Tree of Life Between
 Opisthokonts and All Other Lineages". Sequences and alignments were
 produced using the phylogenomic pipeline PhyloToL, which contains a taxon-
 and gene-rich database (including eukaryotes, archaea, and bacteria).
 These data were then used for 1) assessing the root of the eukaryotes and
 2) for comparison with other previously published hypotheses. In both
 cases, we used the species tree - gene tree reconciliation tool iGTP. We
 also did a comparison of hypotheses using the likelihood-based tool
 SpeciesRax <strong>iGTP</strong>
 <em>Input data</em> These data are
 divided into four datasets based on taxa selection. For dataset SEL+, taxa
 were selected based on their taxonomy; for RAN+, taxa were selected
 randomly among the major eukaryotic clades Opisthokonta, Amoebozoa,
 Archaeplastida, Excavata, SAR, and some orphan lineages. Datasets SEL- and
 RAN- are the same as SEL+ and RAN+, but exclude microsporidians in order
 to account for and avoid long-branch attraction due to
 microsporidians' fast-evolutionary rates. We chose the gene families
 that contain at least 25 taxa representing at least four of the five major
 eukaryotic clades. Additionally, at least 2 of the major clades had to
 contain at least 2 minor clades (e.g. Glaucophytes and Rhodophyta are
 minor clades in the major clade Archaeplastida). In a pilot analysis, we
 produced an alignment and a phylogenetic tree for each gene family using
 the default settings of a previous version of PhyloToL (GUIDANCE V1.3.1
 sequence cutoff = 0.3 and column cutoff = 0.4; RAxML quick tree with model
 PROTGAMMALG and no bootstraps). Then, we kept the gene families that are
 exclusive of eukaryotes or the ones in which eukaryotes were monophyletic.
 From a total of 3,002 gene families that met our criteria, 2786 passed the
 initial steps of PhyloToL when including only the data from the dataset
 SEL+. These 2,786 gene families were used for further analyses with all
 datasets. MSAs were produced with PhyloToL (GUIDANCE
 V2.02 sequence cutoff = 0.3, column cutoff = 0.4, number of iterations =
 5; Sela, et al. 2015). The default parameters of PhyloToL include up to
 five iterations of GUIDANCE V2.02 with 10 bootstraps and MAFFT V7 with
 algorithm E-INS-i for less than 200 sequences or “auto” option if more
 than 200 sequences, and maxiterate = 1000. Instead, here we run up to five
 iterations of GUIDANCE with 20 bootstraps and the simple MAFFT algorithm
 FFT-NS-2. Then, we perform an additional GUIDANCE run with 100 bootstraps
 and the default MAFFT parameters for PhyloToL. Gene
 trees were inferred with RAxML v.8.2.4 with 10 ML searches for best-ML
 tree (option "-# 10"), using the rapid hill-climbing algorithm
 (option "-f d") and no bootstrap replicates. The protein
 evolution model used was evaluated during the gene tree inference (option
 "-m PROTCATAUTO") by testing all models available in RAxML (e.g.
 JTT, LG, WAG, etc) with optimization of substitution rates and of
 site-specific evolutionary rates which were categorized into four distinct
 rate categories for greater computational efficiency.
 We ran 100 repetitions of iGTP analyses per dataset. But, given
 the complexity of the datasets and the heuristic nature of some key steps
 of the iGTP algorithm (e.g. gene tree rooting and initial
 starting species tree generation), in a preliminary analysis,
 we faced two systematic challenges with iGTP as the inferred species tree
 was affected by: 1) the order of the leaves in the input unrooted gene
 tree Newick strings (i.e. the input trees were treated as rooted even
 though we specified that they were not); and 2) the input gene order in
 the 100 replicates. Therefore, we randomly shuffled the order of the
 leaves in the unrooted gene trees (keeping the same topology), and
 randomly shuffled the order of the input gene trees in each of the 100
 replicates per dataset. Here we provide the 100 input files generated for
 those iGTP analyses.  <em>Output
 data</em> Here we also share the data generated
 after two analyses: 1) root assessment and 2) hypothesis testing. For the
 former, we allowed iGTP to calculate the more parsimonious root given our
 input files. For the latter, we allowed iGTP to calculate the
 reconciliation cost of the gene trees given the input files and
 constraints in the species trees to reflect previously published root
 hypotheses. The constraints are explained in the README file.
 <strong>SpeciesRax</strong> Since
 we removed LGT and contamination from our dataset using a series of
 filters, we applied the model UndatedDL instead of UndatedDTL, which
 implies that we only took into consideration duplications and losses and
 ignored the transferences. Then, the command used for SpeciesRax
 was... ./generax --families forGeneRax/famFile
 --species-tree forGeneRax/spsTreeAn.newick --strategy SKIP --rec-model
 UndatedDL --per-family-rates --prefix An_DS1 --si-strategy EVAL 
 <em>input </em> Here, we are
 sharing all the necessary files to run SpeciesRax, including the mapping
 file (famFile), the species trees (spsTrees; the best iGTP constrained
 species trees per hypothesis), and their underlying gene trees
 (trees_r) <em>output</em>
 Output folder from SpeciesRax, which includes log files, events
 (duplications, losses) counts, and statistics (i.e., reconciliation
 likelihood values) Note:  * As in
 the iGTP analyses, for the SpeciesRax files, the words Op, Fu, Di, Un, An,
 refer to the five root hypotheses compared: Opisthokonta-others,
 Fungi-others, Discoba-others, Unikonta-Bikonta, and (Ancyromonadida +
 Metamonada)-others, respectively.  * For the SpeciesRax
 analyses, the Un word refers to Ut (Cavalier-Smith 2003).