An Alternative Root

Current Biology 24, 465–470, February 17, 2014 ª2014 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2014.01.036

Report

for the Eukaryote Tree of Life

Ding He,1,2 Omar Fiz-Palacios,1,2 Cheng-Jie Fu,1

Johanna Fehling,1,3 Chun-Chieh Tsai,1

and Sandra L. Baldauf1,*1Program in Systematic Biology, Department of OrganismalBiology, Uppsala University, Norbyvagen 18D, 75236 Uppsala,Sweden

Summary

The root of the eukaryote tree of life defines someof themost

fundamental relationships among species. It is also criticalfor defining the last eukaryote common ancestor (LECA),

the shared heritage of all extant species. The unikont-bikontroot has been the reigning paradigm for eukaryotes for more

than 10 years [1] but is becoming increasingly controversial[2–4]. We developed a carefully vetted data set, consisting of

37 nuclear-encoded proteins of close bacterial ancestry(euBacs) and their closest bacterial relatives, augmented

by deep sequencing of the Acrasis kona (Heterolobosea,Discoba) transcriptome. Phylogenetic analysis of these

data produces a highly robust, fully resolved global phy-logeny of eukaryotes. The tree sorts all examined eukaryotes

into three megagroups and identifies the Discoba, andpotentially its parent taxon Excavata [5], as the sister group

to the bulk of known eukaryote diversity, the proposedNeozoa (Amorphea + Stramenopila+Alveolata+Rhizaria+

Plantae [SARP] [6]). All major alternative hypotheses are

rejected with as little as w50% of the data, and this resolu-tion is unaffected by the presence of fast-evolving alignment

positions or distant outgroup sequences. This ‘‘neozoan-excavate’’ root revises hypotheses of early eukaryote

evolution and highlights the importance of the poorly stud-ied Discoba for understanding the evolution of eukaryotic

diversity and basic cellular processes.

Results

Thirty-Seven Universal euBac ProteinsMultigene phylogenies now assign most known eukaryotesto a few major groups, and these to three proposedmegagroups—Amorphea, Stramenopila+Alveolata+Rhizaria+Plantae (SARP or Diaphoretickes), and Excavata [5]. Howev-er, little effort has been made to explore the eukaryote rootwith multigene phylogeny, and attempts to root the treewith macromolecular characters have given widely differentresults [1, 3, 4, 6]. We sought to develop a multigene dataset tailored to the study of deep eukaryote phylogeny,including a close outgroup to root the tree. Since mitochon-dria or their remnants are unique to eukaryotes and universalamong them [7], universal eukaryotic genes of bacterialorigin (euBacs) should provide one of the closest possibleoutgroups to root the tree.

2These authors contributed equally to this work3Deceased

*Correspondence: sandra.baldauf@ebc.uu.se

Two parallel protocols employing a combination of homolo-gous clustering and phylogenetic screening were used toidentify proteins suitable for deep eukaryote phylogeny (seeSupplemental Experimental Procedures and Figure S1available online). Screening identified genes that appear tobe (1) of bacterial origin, (2) present in the last eukaryote com-mon ancestor (LECA) (universal or nearly universal amongeukaryotes), and (3) with strong phylogenetic signal (out-paralog free and consistent with well-supported eukaryotephylogeny [5]). Of the 281 universal euBac proteins identified,most failed the latter criteria, primarily due to early gene dupli-cation and lineage-specific losses.Thirty-seven euBacs survived all screening protocols, 33 of

which are known or predicted to function in the mitochondrion(Table S1). To increase sampling for Excavata, we sequencedthe Acrasis kona (Heterolobosea, Discoba) transcriptome,yielding a full set of 37 euBac proteins. Outgroup taxa includedthe closest bacterial relatives of the 37 euBacs (Table S2). AlleuBacs in the final data set reproduce eight or more of theten major eukaryote groups represented, and 36 euBacsshow >60% maximum-likelihood bootstrap (mlBP) supportfor six or more of these major groups (Table S4).

A Rooted Phylogeny of EukaryotesPhylogenetic analysis of a concatenation of the 37 euBacprotein sequences produces a highly resolved phylogeny ofeukaryotes (Figure 1). All major nodes are strongly supportedbased on mlBP [8] and Bayesian inference posterior proba-bilities (biPP) [9]. All ten major eukaryote clades representedare reproduced as monophyletic (1.0/100% biPP/mlBP;Figure 1). These major clades are further organized into threemegagroups (1.0/98%–100% biPP/mlBP; Figure 1): Discoba(Excavata), Amorphea (formerly Unikonta), and SA[R]P(Stramenopila+Alveolata+[Rhizaria not represented]+Plantae).Among these, Amorphea and SA[R]P form a clade to theexclusion of Discoba (1.0/90% biPP/mlBP; Figure 1, matrixM). This identifies Discoba, and potentially its parent taxonExcavata, as the sister lineage to the bulk of knowneukaryote diversity (‘‘Neozoa’’ [6]), thus yielding a ‘‘neozoan-excavate’’ root.Amajor concern in a rooted phylogeny is possible artifactual

attraction of long ingroup branches toward the potentially longbranches of a distant outgroup (long-branch attraction, orLBA) [10].We tested the euBac phylogeny for possible sourcesof LBA, particularly signal dependence on fast-evolving align-ment positions and distant outgroup sequences. Removal ofthe fastest-evolving alignment positions (3,017 sites, or 21%of the data set), defined in a tree-independent manner [11],yielded an identical maximum-likelihood tree with strongsupport for the neozoan-excavate root (0.99/92% biPP/mlBP; Figure 1, matrix M0). Sequences with distant bacterialhomologs were identified based on the maximum likelihoodbranch length connecting ingroup and outgroup, correctedfor the number of informative sites (‘‘IOScore,’’ Table S1;also see Experimental Procedures). Masking bacterialsequences for the ten euBacs with IOScores > 1.0 increasesrather than decreases support for the neozoan-excavate root(0.99/100% biPP/mlBP; Figure 1 matrix M00).

Figure 1. A Rooted Phylogeny of Eukaryotes Based on Thirty-Seven Eukaryotic Proteins of Bacterial Ancestry

The tree shown was derived from the full euBac data set (matrix M) by maximum likelihood using RAxML 7.6.6 [8]. Branches are drawn to scale as indicated

by the scale bar. Major groups are indicated by different colors and to the right with brackets and names. Nodal support from maximum-likelihood boot-

strapping (mlBP, above branches) and Bayesian inference posterior probabilities (biPP, below branches) is shown only for nodes that did not receive full

support (100% mlBP and 1.0 biPP). The inferred topologies from different data sets or analyses were identical except where indicated by dashed lines, for

which only the highest support values are shown. Statistical support for the two nodes flanking the root (denoted a and b) is shown separately in the upper

left for analyses with all alignment positions (matrix M), analyses with fast-evolving sites removed (matrix M0), or analyses with bacterial sequences masked

for the ten euBac proteins with IOScore > 1.0 (matrix M00).

Current Biology Vol 24 No 4466

Given three strongly supported eukaryote megagroups,there are three possible alternative hypotheses for the eukar-yote root with these data: H1, Discoba sister to Amorphea +SA[R]P (neozoan-excavate root; Figure 1); H2, Amorphea sisterto Discoba + SA[R]P (unikont-bikont root) [1]; and H3, SA[R]Psister to Discoba + Amorphea [12]. These hypotheses weretested against each other using the approximately unbiased(AU) test [13] with repeat sampling of matrices of increasingsize. This cumulative AU approach shows increasing supportfor H1, until H2 and H3 are rejected with R22 and R32randomly sampled euBacs, respectively (Figure 2A).Whenout-group sequencesaremasked for euBacswith an IOScore>1.0,H2 and H3 are rejected with R12 and R17 randomly sampledeuBacs, respectively (Figure 2B). The same protocol rejectsthe two main alternatives for discobid paraphyly with R12randomly sampled euBacs (data not shown).

Contradictory DataTheonly other recent test of the eukaryote root usingmultigenephylogeny employed 42 mitochondrial proteins of a-proteo-bacterial ancestry (amitoP proteins [14]). That study reported

strong support for a unikont-bikont root, particularly with theremoval of fast-evolving alignment positions defined in atree-dependent manner. Only 13 proteins are shared betweenthe euBac and amitoP data set (Table S1), indicating that 29amitoP proteins failed some euBac vetting criteria. The globalcongruence test ofConclustador [15] also indicates substantialincongruent signal within the amitoP (but not the euBac) data,particularly with respect to the phylogenetic positions of somediscobids. Therefore, we tested the effect of removingsubsets of Discoba on amitoP and euBac phylogeny.Using all possible combinations of the three major divisions

of Discoba (Jakobida, Euglenozoa, Heterolobosea [5]), theeuBac data retrieve the neozoan-excavate root and alleukaryote supergroups with moderate to strongmlBP support(Table 1). However, the amitoP data fail to reproduce theunikont-bikont root and several eukaryote supergroups withnearly all combinations of discobids (Table 1). Most strikinglywith only Jakobida represented, the amitoP data stronglyplace Discoba in SA[R]P together with Plantae (95% mlBP;Table 1, amitoP data set B2), and with only Heterolobosea rep-resented, Discoba are strongly placed in Amorphea together

Figure 2. Evolution of Phylogenetic Signal in the euBac Data Set

Matrices of increasing size (numbers of euBacs) were built by 500 rounds of

random addition permatrix size class in increments of five (from 2 to 32) with

the full euBac data set (A) and with bacterial sequences masked for the ten

highest IOScore euBacs (IOScore > 1.0) (B). Three hypotheses for the

eukaryote root were compared for each matrix in each size class using

the approximately unbiased (AU) test [13] as follows: H1 (blue), Discoba sis-

ter to SA[R]P + Amorphea (neozoan-excavate root; Figure 1); H2 (red),

Amorphea sister to Discoba + SA[R]P (unikont-bikont root; [1]); and H3

(green), SA[R]P sister to Amorphea + Discoba. Probabilities (boxplots) for

the three hypotheses are plotted (y axis) against matrix size class (number

of euBacs, x axis). The gray area at the bottomof each graph indicates rejec-

tion of the given hypothesis at p < 0.05. Boxes were drawn from the first to

the third quartile of the data, with median values indicated by the horizontal

lines in the boxes. Dashed lines extending out of the boxes indicate the

maximum and minimum values excluding the outliers.

The Eukaryote Root467

with Amoebozoa (99% mlBP; amitoP data set B4). Such con-flicting signals strongly suggest that the genes encodingsome discobid amitoP proteins have been replaced by hori-zontal gene transfer (HGT) with homologs from distantlyrelated taxa. Thus, the amitoP data appear to contain threeopposing phylogenetic signals, one uniting Discoba and twoplacing subsets of Discoba on opposite sides of the tree.

Discussion

Strong Support for an Alternative Eukaryote RootOf the three eukaryote megagroups identified here (Figure 1),Amorphea and Discoba are well established [5], while SARPis less consistently recovered [16–19]. The strong evidence

here for a monophyletic SA[R]P (Figure 1) may be due to theavoidance of large amounts of missing data and/or the exclu-sion of difficult-to-resolve (rogue) taxa [20]. Although the cur-rent euBac data set is very taxonomically broad, there are stillmany missing taxa, some of which may be found to branchdeep to Discoba or between Discoba and Neozoa. Neverthe-less, no addition of taxa can change the fundamental relation-ship identified here, i.e., that Amorphea and SARP are moreclosely related to each other than either is to Discoba.

Missing TaxaA number of potentially important taxa are missing from thepresent study because of inadequate available sequencedata or poor taxon sampling (singleton taxa). The possibleimpact of some of these taxa on the root was assessed witha preliminary expanded euBac phylogeny (Figure S2). Thisphylogeny confirms placement of the single available apuso-zoan (Thecamonas) as sister to Opisthokonts [14] and the sin-gle available rhizarian (Bigelowellia) within SARP (100%mlBP;[16, 17]), tentatively as sister lineage to Alveolata (73% mlBP).The single available cryptophyte (Guillardia) and haptophyte(Emiliania) form a surprisingly strong clade (100% mlBP [16],but see [18, 19, 21]), suggesting the possible integrity of atleast part of the proposed Hacrobia [5]. This clade furthershows some affinity for Archaeplastida (71% mlBP), althoughits presence appears to blur the phylogenetic signal for amonophyletic Archaeplastida (82% mlBP), which is otherwisevery strong (100% mlBP; Figure S2). The very early-divergingdiscobid Andalucia [22] is also confidently placed within Dis-coba, despite 90% missing data (91% mlBP), and tentativelyas the earliest branch of Jakobida (71% mlBP; [23]). Mostimportantly, inclusion of this collection of incomplete and/orsingleton taxa does not diminish support for the neozoan-excavate root (91%–93% mlBP; Figure S2).More important missing taxa with regard to the eukaryote

root are currently unassigned mitochondriate species withexcavate morphology—Malawimonas [24], Collodictyon [19],and possibly Tsukubamonas [25]. However, the most impor-tant missing taxa are the remaining Excavata, the Fornicataand Preaxostyla [26], referred to here as amitochondriateexcavates (AME). A monophyletic Excavata (AME + Discoba)has yet to be tested in a rooted multigene phylogeny. If theeukaryote root lies within Excavata (paraphyletic excavates),this would mean that the morphologically complex excavatedfeeding groove [26] is not a synapomorphy for Excavata butrather an ancestral eukaryotic trait. However, since all knownAME taxa lack aerobic mitochondria, they are excluded fromthe 37-euBac data set.

Why Is the euBac Phylogeny Not Rooted with

a-Proteobacteria?Thirty-three of the euBacs are predicted to be mitochondriallytargeted (Table S1), indicating that the euBac data consistlargely of components of the LECA mitochondrial proteome.Given that mitochondrially encoded genes often show astrong a-proteobacterial affinity [27], it may seem logicalthat a tree of nuclear-encoded mitochondrial proteins shouldbe rooted with a-proteobacteria. However, only a smallfraction of nuclear-encoded mitochondrial proteins trace toa-proteobacteria (e.g., only 10% in yeast [27]), whereas therest show either a dispersed bacterial signature or are uniqueto eukaryotes [27–29]. In the process of assembling the euBacdata set, we effectively tested the hypothesis that a-proteo-bacterial euBacs (amitoP proteins) are suitable for deep

Table 1. Taxon Jackknife Analysis of euBac and amitoP Data Sets

Data Set Discoba Divisions Included

Major Eukaryote Groups Root

Opi Amb AMR Pla SA[R] SA[R]P Dis uni-bik neo-exc

euBac (76 taxa) A1 Jak, Htr, Eug 100 100 100 100 97 99 100 – 90

A2 Jak 100 100 99 100 100 94 NA – 97

A3 Jak, Htr 100 100 96 100 99 100 100 – 84

A4 Htr 100 100 65 100 98 86 NA – 65

A5 Eug 100 100 100 100 56 51 NA – 57

A6 Htr, Eug 100 100 92 100 87 89 100 – 85

A7 Jak, Eug 100 100 100 100 89 89 62 – 86

amitoP (47 taxa) B1 Jak, Htr, Eug 100 100 257*1 100 76 273*2 XX XX –

B2 Jak 100 100 99 100 94 295*2 NA XX –

B3 Jak, Htr 100 100 281*3 100 91 286*2 XX XX –

B4 Htr 100 100 299*3 100 80 96 NA XX –

B5 Eug 100 100 78 100 266*4 266*4 XX XX –

B6 Htr, Eug 100 100 279*1 100 58 80 100 XX –

B7 Jak, Eug 100 100 85 100 65 31 46 71 –

B8 Jak, Htr, Eug 100 100 257*1 100 86 239*2 XX XX –

Numbers correspond to maximum-likelihood bootstrapping support for major groups listed across the top and two alternative eukaryote roots listed at the

far right, expressed as support for a unikont-bikont (SA[R]P+Dis) or neozoan-excavate root (SA[R]P+AMR). Major groups scored are Opisthokonta (Opi),

Amoebozoa (Amb), Amorphea (AMR), Viridiplantae (Pla), Stramenopila+Alveolata (SA[R]), SA[R]+Viridiplantae (SA[R]P), and Discoba (Dis). Data

sets were analyzed with all possible combinations of the three major divisions of Discoba: Euglenozoa (Eug), Jakobida (Jak), and Heterolobosea (Htr).

For the amitoP data [14], a 47-taxon subset excluding potential rogue taxa was used to maximize signal, except for amitoP data set B8, for which all

54 taxa were included. XX indicates nonmonophyletic Discoba due to subsets being dispersed across the tree, as indicated by minus signs and

asterisks (*), with superscript numbers indicating the supported alternative groups: 1(Htr+Eug+Amb), 2(Jak+Pla), 3(Htr+Amb), 4(Eug+Ciliophora). NA, not

applicable.

Current Biology Vol 24 No 4468

eukaryote phylogeny (Figure S1), and we found that few ofthem are.

We find instead that nuclear-encoded mitochondrialgenes with robust phylogenetic signal trace to a variety ofbacteria, albeit mostly proteobacteria. This is consistent witha mosaic mitochondrial progenitor with some genes origi-nating by HGT from various bacteria or possibly even replacedearly within eukaryotes after mitochondrial acquisition [28, 29].Mitochondrial gene origins are undoubtedly further obscuredby 1–1.5 billion years of HGT among bacteria since endosym-biosis [28, 29]. Thus, it is highly unlikely that the mitochondrialprogenitor genome has survived intact in any single organism,if it ever fully existed as such in the first place.

Nevertheless, regardless of their ancient history, the 37euBacs examined here show strong evidence of havinginhabited the early mitochondrial or mitochondrial progenitorproteome. This hypothetical proteome is partially recon-structed here, corresponding to the last common node sharedby all outgroup taxa (last common outgroup ancestor, orLCOA), which in turn is the closest outgroup to the root, thelast common node shared by all ingroup taxa (LECA). Regard-less of any possible mixed history within the outgroup, thegenes used here appear to have followed a single evolutionarytrajectory fromtheoutgroupancestor node (LCOA) until thefirstmajor radiation of extant eukaryotes (LECA). Thus, the root issubtended here by a long history of simple phylogeny. It shouldbe noted that although the euBac phylogeny is rooted with acomposite outgroup, this is in fact the case for any phylogenyrooted with multiple outgroup taxa. Furthermore, compositeoutgroups give more accurate roots, at least with discretedata methods such as are used here, as they more accuratelyreconstruct the ancestor of the root (LCOA, Figure 1; [30]).

Decoding Conflicting Signals for the Eukaryote RootOur analysis of a data set consisting of all eukaryote proteinswith a strong a-proteobacterial affinity (amitoP data [14])shows strong signatures of HGT among major groups of

eukaryotes. Among other things, this emphasizes the impor-tance of selecting multigene data based on empiricalperformance [31]. In fact, strong mlBP support for the uni-kont-bikont root from the amitoP data was found only afterremoving fast-evolving sites defined using tree topologiesnot recovered by the data, i.e., Heterolobosea + Euglenozoa +Plantae,whichalsoexcludes the rootwe recover here (Figure1;[14]). Interestingly, this topology also excludes one paraphy-letic signal (Heterolobosea + Amoebozoa) but not the other(Jakobida + Plantae), the latter in itself strongly promoting anExcavata+SARP group, i.e., a unikont-bikont root.Most recent attempts to root the eukaryote tree have

focused on macromolecular characters in an attempt to avoidpotential LBA artifacts. However, recent studies reveal sur-prisingly high levels of homoplasy (independent origin andreversal) in such characters [32–34]. For example, the uni-kont-bikont root originally relied largely on an apparently raregene fusion [1], but recent analyses identify multiple fusionsand fissions of these genes within fungi alone [35]. A possibleeuglenozoan [6] root based on their unusual molecularfeatures is rejected here by strong support for monophyleticDiscoba in a rooted tree (Figure 1), suggesting that these fea-tures are probably either retained primitive characters oreuglenozoan-specific inventions. A recently proposed ‘‘photo-synthetic-nonphotosynthetic’’ root based on rare amino acidsubstitutions [4] is also strongly rejected here (Figures 1 and2) and by abundant evidence that plastids spread acrosseukaryotes by horizontal transfer [36]. Finally, an opisthokontor possibly fungal root for eukaryotes recovered byminimizinghypothesized ancient gene duplications and losses [3] is con-tradicted by evidence of repeated independent gene familyexpansion and contraction throughout eukaryotes (see aboveand e.g. [37]).

Implications of a Neozoan-Excavate Root

The neozoan-excavate root identifies Discoba as belonging toa major ancient lineage that has been evolving independently

The Eukaryote Root469

since the first speciation events of extant eukaryotes.Expressed in terms of more familiar taxa, the neozoan-exca-vate root means that Discoba diverged from the main line ofeukaryote descent well before the origin of the separate line-ages that eventually gave rise to plants, animals, and fungi.Nonetheless, other than the well-studied parasites Trypano-soma and Leishmania, most molecular information on disco-bids concerns their mitochondrial genomes (mtDNA). Theserange from the large, gene-rich bacterial-like mtDNAs of jako-bids [23] to the highly fragmented and/or massively editedmtDNAs of Euglenozoa [38]. This limited molecular samplingsuggests that Discoba could harbor a further wealth of biolog-ical novelty.

The neozoan-excavate root also emphasizes the impor-tance of Discoba, and probably also AME, for understandingearly eukaryote evolution. Extensive similarities have beenidentified between Neozoa and the only fully sequencedfree-living discobid, Naegleria gruberi [39]. However,Naegleria is still only a single data point in the vast stretch ofevolutionary time separating Discoba and the bulk of knowneukaryotes. Further data are clearly needed from a broadersampling of Excavata in order to distinguish possibly uniquefeatures of Naegleria from the common heritage of extanteukaryotes.

Experimental Procedures

Acrasis kona Protein Sequences

A cDNA library was constructed from total RNA extracted from a

laboratory-grown culture of Acrasis kona ATCC strain MYA-3509 (formerly

Acrasis rosea [40]) and sequenced on a 454 GS FLX Titanium

platform. For details of cell culture, RNA extraction, sequencing, assembly,

and transcript screening, please see the Supplemental Experimental

Procedures.

Identification of Universal euBacs

Two separate pipelines were used to identify euBacs and determine their

suitability for deep eukaryote phylogeny (Figure S1). Both protocols used

the predicted proteomes from 12 completely sequenced eukaryote

genomes as a starting point (Supplemental Experimental Procedures; Fig-

ure S1). The 12 included at least one representative each from five of the

six currently recognized supergroups of eukaryotes (Opisthokonta,

Amoebozoa, Archaeplastida, Chromalveolata, and Excavata; [5]), as sub-

stantial genomic data were unavailable from Rhizaria during these analyses

(Table S2).

Final euBac Data Set Assembly

Eukaryotic sequenceswere taken primarily from the predicted proteomes of

completely sequenced genomes (Table S2). Missing entries and data from

taxa without complete genome sequences were obtained by BLASTp

searches of the NCBI nr or Expressed Sequence Tags (EST) databases or

other publically available genome data, as needed. Some sequences were

also assembled by hand from EST data, particularly for jakobids. Whenever

possible, sequences were taken from a single target genome (Table S2), but

for many taxa one or two genes could be found only in related taxa. In these

cases, sequences were taken from the most closely related taxon possible,

which was always in the same genus. Bacterial sequences were retrieved

separately using a taxonomic hierarchy protocol (Supplemental Experi-

mental Procedures; Table S3). During assembly, the data set was checked

regularly and, especially when complete, by single-gene control trees to

assure that all sequences were orthologs. The final 37 trimmed alignments

were assembled by hand into a single interleaved full data matrix M. Two

modified versions of this matrix were generated by (1) removing the

fastest-evolving alignment positions (matrix M0; see below) and (2) removing

bacterial sequences for the ten proteins with IOScores > 1.0 (matrix M00; seebelow and Table S1).

Phylogenetic Analyses

Maximum-likelihood bootstrap analyses consisted of 100 rapid bootstrap

replicates (mlBP) performed using theWAG amino acid substitution models

corrected for observed amino acid frequencies and among-site rate varia-

tion using a four-discrete-category gamma distribution as implemented in

RAxML 7.6.6 [8]. Bayesian inference analyses allowing for site-heteroge-

neous amino acid replacement processes and rate variation among sites

and among lineages utilized the CAT+G4+covarion model with constant

sites removed. These analyses were run on two independent chains using

PhyloBayes 3.3 [9]. The convergence of two root-defining nodes (a and b;

Figure 1) among chains was diagnosed based on a comparison of the

frequency of all bipartitions after discarding w50% of the cycles as burn-

in (maxdiff < 0.1). All analyses were run on a local cluster, the Uppsala

Multidisciplinary Center for Advanced Computational Science (UPPMAX,

http://www.uppmax.uu.se), BioPortal (http://www.mn.uio.no/ibv/bioportal/

index.html), or the Cyberinfrastructure for Phylogenetic Research (CIPRES,

http://www.phylo.org; [41]), depending on availability.

Vetting Sources of Artifacts and Incongruence

The corrected distances between ingroup and outgroup (IOScore) were

calculated from individual maximum-likelihood control trees [8]. The value

corresponds to the length of the branch connecting ingroup and outgroup

(dIO), divided by the number of informative sites (InfSites), multiplied by

1,000 for ease of interpretation [(dIO/InfSites) 3 1,000)]. The value for

InfSites was calculated with PAUP* [42] under the heuristic search function

and corresponds to all sites with at least two different states shared by a

minimum of two taxa each. Fast-evolving sites were determined in a tree-

independent manner using the program TIGER [11] with default settings.

Sites were assigned into ten rate bins. Topology-based congruent tests

were run using Conclustador with maximum-likelihood bootstrap method

as described in [15].

Cumulative AU Test

Alternative hypotheses were tested using the approximately unbiased (AU)

test [13] on matrices of increasing size from 2 to 32 in increments of 5 due to

computational constraints. Matrices were built by random sampling without

replacement of the 37-euBac protein data set. Sampling consisted of 500

independent replicates for each matrix size class, yielding a total of 3,500

unique matrices, with any identical replicates automatically deleted. AU

tests for alternative phylogenetic hypotheses were performed on each

matrix under the WAG substitution model with 100,000 bootstrap replicates

using the program TREEFINDER [43]. The median p values for each

hypothesis were then calculated from the p values for all samples in each

matrix size class.

Accession Numbers

Deduced amino acids sequences for the 37 euBacs from Acrasis kona have

been deposited in GenBank with accession numbers JL968957–JL968959,

JL968961–JL968982, JL968984, and JL968986–JL968996. The 37-euBac

protein data matrices have been deposited in TreeBASE (http://treebase.

org/; Study ID S14693).

Supplemental Information

Supplemental Information includes two figures, four tables, and Supple-

mental Experimental Procedures and can be found with this article online

at http://dx.doi.org/10.1016/j.cub.2014.01.036.

Acknowledgments

We thank P. Ajawatanawong, E. Lundin, and L. Ponnuswamy for assistance

in writing scripts; J.W. Leigh for assistance in running Conclustador; and M.

Brown and F. Spiegel for kindly providing Acrasis kona cultures. We also

thank the Joint Genome Institute, Protist Database Project, andWashington

University Genome Institute for the use of unpublished sequences and

UPPMAX, BioPortal, and CIPRES for the use of computational resources.

This work was supported by grants from the Swedish Research Council

(Vetenskapradet). This paper is dedicated to the memory of Dr. Johanna

Fehling (1979–2011).

Received: November 4, 2013

Revised: December 12, 2013

Accepted: January 16, 2014

Published: February 6, 2014

Current Biology Vol 24 No 4470

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