Evolutionary Relationships of Apusomonads Inferred from Taxon-Rich Analyses of 6 Nuclear Encoded...

and Evolution. All rights reserved. For permissions, please e-mail: [email protected] The Author 2006. Published by Oxford University Press on behalf of the Society for Molecular Biology

Evolutionary relationships of apusomonads inferred from taxon-rich analyses of six

nuclear-encoded genes

Research Article

Eunsoo Kim1, Alastair G. B. Simpson2, Linda E. Graham1

1Department of Botany, University of Wisconsin-Madison, 132 Birge Hall, 430 Lincoln

Dr., Madison, WI 53706, USA

2Department of Biology, Dalhousie University, Halifax. N.S., B3H 4J1, Canada

Correspondence to: Eunsoo Kim, E-mail: [email protected], Phone: 608-262-0657

Fax: 608-262-7509

Contact information: Eunsoo Kim, Address: Department of Botany, University of

Wisconsin-Madison, 132 Birge Hall, 430 Lincoln Dr., Madison, WI 53706, USA

Running head: Phylogenetics of apusomonads

Key words: Apusomonas, Apusomonadidae, Opisthokonta, Amoebozoa, protists,

evolution

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ABSTRACT

The phylogenetic relationships of the biflagellate protist group Apusomonadidae have

been unclear despite the availability of some molecular data. We analyzed sequences

from six nuclear encoded genes—SSU rRNA, LSU rRNA, α-tubulin, β-tubulin, actin,

and Hsp90—to infer the phylogenetic position of Apusomonas proboscidea Aléxéieff

1924. To increase the taxon richness of the study, we also obtained new sequences from

representatives of several other major eukaryotic groups: Chrysochromulina sp. NIES

1333 (Haptophyta), Cyanophora paradoxa (Glaucophyta), Goniomonas truncata

(Cryptophyceae), Leucocryptos marina (Kathablepharidae), Mesostigma viride

(Streptophyta, Viridiplantae), Peridinium limbatum (Alveolata), Pterosperma cristatum

(Prasinophytae, Viridiplantae), Synura sphagnicola (Stramenopiles), and

Thaumatomonas sp. (Rhizaria). In most individual gene phylogenies, Apusomonas

branched close to either of two related taxa - Opisthokonta (including animals, fungi, and

choanoflagellates) or Amoebozoa. Combined analyses of all four protein-coding genes,

or all six studied genes strongly supported the hypothesis that Apusomonadidae is closely

related to Opisthokonta (or to all other eukaryotic groups except Opisthokonta, depending

the position of the eukaryotic root). Alternative hypotheses were rejected in AU tests at

the 5% level. However, the strong phylogenetic signal supporting a specific affiliation

between Apusomonadidae and Opisthokonta largely originated from the α-tubulin data.

If α-tubulin is not considered, topologies in which Apusomonadidae are sister to

Opisthokonta or are sister to Amoebozoa were more or less equally supported. One

current model for deep eukaryotic evolution holds that eukaryotes are divided into

primary ‘unikont’ and ‘bikont’ clades and are descended from a ‘uniflagellate’ common

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ancestor. Together with other information, our data suggest instead that ‘unikonts’

(=Opisthokonta and Amoebozoa) are not strictly monophyletic and are descended from

biflagellate ancestors.



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INTRODUCTION

Our understanding of eukaryotic phylogeny has improved in recent years as the

result of increasing sequence data from diverse taxonomic groups. For example, a

molecular gene analysis revealed that many morphologically diverse protists form a

superclade known as Rhizaria (Nikolaev et al. 2004). Most eukaryotes are now placed

into one of about 15 major eukaryotic lineages whose monophyly is generally

undisputed. Some authors reduce these lineages to six ‘supergroups’ (Simpson and Roger

2004; Adl et al. 2005; Keeling et al. 2005), although the monophyly of some of these

supergroups is currently under debate. More contentiously still, it has been proposed that

all major lineages fall into just two primary clades – ‘unikonts’ and ‘bikonts’ (Stechmann

and Cavalier-Smith 2003, Richards and Cavalier-Smith, 2005) However, a number of

protist taxa cannot be assigned unambiguously to any of these major eukaryotic lineages,

despite the presence of electron microscopical data and at least some sequence data

(Simpson and Roger 2004; Adl et al. 2005). These unassigned taxa are pivotal for

understanding eukaryotic diversification and for testing macroevolutionary hypotheses

such as the unikont/bikont bifurcation (Stechmann and Cavalier-Smith 2003b; Cavalier-

Smith, Chao, Oates 2004). Apusomonadidae, the focus of our study, is one such group.

The Apusomonadidae (‘apusomonads’) are a group of free-living heterotrophic

biflagellates consisting of two genera—Amastigomonas and Apusomonas. Apusomonads

glide along surfaces and feed on bacteria, which are usually engulfed using ventral

pseudopodia (Vickerman, Darbyshire, Ogden 1974). Their cells are covered with a

thickened submembranous ‘theca’ except in the ventral feeding region (Molina and

Nerad 1991). The cells possess two heterodynamic flagella: one anteriorly-directed and



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one posteriorly-oriented (Vickerman, Darbyshire, Ogden 1974). The anterior flagellum is

encased by a membranous sleeve, a trait that is a synapomorphic feature for

Apusomonadidae (Patterson 2000a). The two basal bodies are inserted at right angles and

three rootlets are associated with these (Molina and Nerad 1991). One rootlet is a

multilayered structure (MLS), while two other rootlets, each consisting of 3–5 and 5–12

microtubules, underline each side of the ventral groove (Karpoff and Zhukov 1986;

Molina and Nerad 1991). Mitochondria have tubular cristae (Karpoff and Zhukov 1986;

Molina and Nerad 1991). A distinguishing feature of Apusomonas is the mastigophore, a

long extension of the two ventral grooves, from which the two flagella originate

(Patterson 2000b). SSU rRNA phylogenes clearly established the monophyly of

Apusomonadidae (Cavalier-Smith and Chao 2003b).

Thus far, two nuclear encoded genes—SSU rRNA and heat shock protein (Hsp)

90—have been used to infer the phylogenetic relationships of Apusomonadidae

(Cavalier-Smith and Chao 1995; Stechmann and Cavalier-Smith 2003). Based on SSU

rRNA gene phylogenies, Cavalier-Smith and Chao (1995) initially suggested that

Apusomonas is closely related to Opisthokonta, the clade that includes animals, fungi,

and choanoflagellates, with this hypothesis receiving moderate bootstrap support. The

same relationship was also recovered in later SSU rRNA studies, but with lower

bootstrap support (eg. Atkins, McArthur, Teske 2000; Fig 1 in Cavalier-Smith and Chao

2003b; Fig 4 in Cavalier-Smith, Chao, Oates 2004). However, other analyses have not

recovered a close association between Apusomonadidae and Opisthokonta (eg. Fig 2 in

Cavalier-Smith 2002; Simpson et al. 2002; Fig 2 in Cavalier-Smith, Chao, Oates, 2004;

Berney, Fahrni, Pawlowski 2004). Analyses based on Hsp90 gene sequences including



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that of Amastigomonas marina did not strongly support any particular phylogenetic

position for apusomonads (Stechmann and Cavalier-Smith 2003a). Furthermore, the

dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene fusion was identified in

Amastigomonas debruynei, suggesting a placement of apuosomonads in the large ‘bikont’

clade supposedly identified by this fusion character, and therefore remote from the

‘unikont’ clade (Opisthokonts and Amoebozoa), whose members lack this gene fusion

(Stechmann and Cavalier-Smith 2002; 2003b).

In this study, we sequenced five nuclear encoded genes from Apusomonas

proboscidea (LSU rRNA, α-tubulin, β-tubulin, actin, and Hsp90), and performed various

phylogenetic analyses with the goal of resolving the phylogenetic position of

Apusomonadidae. We also analyzed existing SSU rRNA gene data from apusomonads.

New sequences were also obtained from several distantly related protists in known

eukaryotic groups, in order to increase the taxon richness of our study. Organisms

included were Chrysochromulina sp. NIES 1333 (Haptophyta), Cyanophora paradoxa

(Glaucophyta), Goniomonas truncata (Cryptophyceae), Leucocryptos marina

(Kathablepharidae), Mesostigma viride (Streptophyta, Viridiplantae), Pterosperma

cristatum (Prasinophytae, Viridiplantae), Peridinium limbatum (Alveolata), Synura

sphagnicola (Stramenopiles), and Thaumatomonas sp. (Rhizaria). We carefully chose

taxa with sequences that are not particularly long-branched and/or that are early-

diverging members of some major eukaryotic lineages, and performed both Maximum

Likelihood (ML) and Bayesian analyses of individual and combined gene data sets.



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MATERIALS AND METHODS

Cultures

Apusomonas proboscidea Aléxéieff (CCAP 1905/1) was purchased from the

Culture Collection of Algae and Protozoa (CCAP), Argyll, Scotland. Chrysochromulina

sp. (NIES 1333), Leucocryptos marina (Braarud) Butcher (NIES 1335), Mesostigma

viride Lauterborn (NIES 296) and Pterosperma cristatum Schiller (NIES 936) were

obtained from the Microbial Culture Collection at the National Institute for

Environmental Studies (MCC NIES), Ibaraki, Japan. Cyanophora paradoxa Korshikov

and Glaucocystis nostochinearum Itzigsohn var. nostochinearum were obtained from

Carolina Biological Supply Company, Burlington, North Carolina. Goniomonas truncata

(Fresenius) Stein, Peridinium limbatum (Stokes) Lemmermann, Synura sphagnicola

(Korshikov) Korshikov, and Thaumatomonas sp., were cultured from various lakes in

Wisconsin, USA following single cell isolation (Stein 1975) and were maintained in

appropriate culture media—in most cases, a mixture of sterilized filtered lake water and

soil extract. Cultures were maintained at 15° C. Cultures were identified both by light

microscopic features and SSU rRNA gene sequences. The isolate identified as

Thaumatomonas sp. had SSU rRNA gene sequence 99.8% similar to database sequences

for Thaumatomonas sp. (SA), based on an NCBI BLAST sequence similarity search.

DNA/RNA Preparation

Cells were concentrated by centrifugation and genomic DNA was extracted using

the DNeasy Plant Mini Kit (Qiagen, Valencia, CA), according to the manufacturer’s

suggested protocols. Cells of Glaucocystis, Mesostigma, Peridinium, Pterosperma, and



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Synura were disrupted in liquid nitrogen with a plastic pestle. Vigorous vortexing in the

lysis solution was sufficient for colorless protist cells. In some cases, use of degenerate

PCR primers for amplifications of protein-coding genes from genomic DNA resulted in

multiple bands presumably representing non-specific amplification, and RT-PCR was

necessary. Total RNA was purified from Apusomonas, Chrysochromulina, Goniomonas,

Leucocryptos, and Thaumatomonas, using the RNeasy Mini Kit (Qiagen, Valencia, CA),

and cDNA was synthesized from oligo dT primers using the Access RT-PCR kit

(Promega, Madison, WI).

PCR Amplification, Cloning, and Sequencing

PCR primers for amplifying SSU rRNA, LSU rRNA, α-tubulin, β-tubulin, actin,

and Hsp90 gene sequences were designed based on sequence alignments as well as

previous studies (Table 1) (Simpson, Lukes, Roger 2002; Simpson, Inagaki, Roger 2006).

For some protein coding genes, a two-step nested PCR technique was applied. The

standard 50 µl reaction mixture consisted of 2.5 unit of Takara Ex Taq (Takara, Tokyo),

1X Ex Taq buffer, 0.2 mM of each dNTP, 0.6 µM of each primer, and 5% glycerol.

When PCR primers were degenerate at several positions, the standard PCR cyclic

reactions consisted of a denaturation step at 95°C for 3 min; 13 cycles of 1 min at 95°C, 1

min at 58°C (1°C decrease each cycle), and 1.5 min at 68°C; 20 cycles of 30 sec at 94°C,

1 min at 45°C, and 1.5 min at 68°C; and a final 10 min at 68°C. For SSU rRNA and LSU

rRNA gene amplifications, the standard PCR cyclic reactions consisted of a denaturation

step at 95°C for 3 min; 30 cycles of 1 min at 95°C, 1 min at 45°C, and 1~3 min at 72°C;

and a final 15 min at 72°C. For Hsp90 from Apusomonas, a 330bp fragment near the 5’



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end of the coding region was amplified by nested PCR and used to design an exact-match

primer. A near-complete coding region was then amplified by a nested PCR with this

exact-match primer. The 20 µl reaction mixtures consisted of 1 unit of Taq polymerase

(Sigma-Aldrich, St. Louis, MO), 1X buffer (1.5mM MgCl2), 0.2 mM of each dNTP and

1 µM of each primer. The cycling for the final PCR consisted of a denaturation step at

94°C for 2 min; 35 cycles of 20s at 94°C, 1 min at 54°C, and 2.5 min at 72°C; and a final

5 min at 72°C. PCR-amplified fragments were either gel-purified, or were cleaned using

Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI), and then were

cloned into pCR 4-TOPO or pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA) or

pGEM®-T Easy vector (Promega, Madison, WI). Plasmids were isolated from multiple

positive bacterial clones using the QIAquick Miniprep Kit (Qiagen, Valencia, CA) or

Sigma miniprep kit (Sigma-Aldrich, St. Louis, MO). Multiple clones were partially

sequenced, and at least one clone from each reaction was selected for complete

sequencing. To eliminate the possibility of contamination, the identities of all gene

sequences were verified as described in online Supplementary Material (Method S1).

GenBank accession numbers of new sequences obtained in this study are listed in online

Supplementary Material (Table S1).

Sequence Alignments

Protein coding genes were translated to amino acids, which were manually

aligned using MacClade ver. 4.05 (Maddison and Maddison 2001). For the alignment of

SSU rRNA genes, CLUSTAL X (Thompson et al. 1997) was used to produce an

approximate alignment, after which the sequences were aligned more accurately based on



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eukaryotic SSU rRNA secondary structure models (Wuys et al. 2002) from the European

database. Yves Van de Peer (Ghent University) kindly provided the LSU rRNA gene

sequence alignment used in a previous study (Ben Ali et al. 2001), which incorporated

both primary and secondary structure information. We added our new sequences and

other sequences available from GenBank. Ambiguously aligned positions were excluded

for all analyses. Initial individual gene alignments included more sequences than the final

versions. This allowed detection of possible paralogy and selection of short-branched

homologous copies. Through various preliminary phylogenetic analyses, mostly using

neighbor joining and maximum parsimony methods, long-branched or potentially non-

orthologous sequences were identified and excluded. For example, sequences of most

animals and embryophytes, and some fungi were removed from our α-tubulin, β-tubulin,

or actin gene alignments because multiple paralogs were present in these multicellular

organisms. Multiple gene copies, present in some of the included taxa, were closely

related to each other, to the exclusion of all other sequences analyzed. We carefully chose

taxa included in the final alignments to increase overall taxonomic representation of the

study, yet minimize potential problems associated with long-branch attraction artifacts

(Philippe 2000). In some cases, gene sequences from closely related taxa were

concatenated for the combined gene analyses. No alignment position or taxon was

included in the analysis if more than 20% of the total data were missing. In addition, in

the combined gene alignments, no taxon was included if more than 40% of the individual

gene data were missing. Sequence alignments are deposited at TreeBASE

(www.treebase.org).



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Because we restricted sequence length and percentage of included positions

across taxa, missing data did not exceed 3% in any one alignment. Only 1.6% of

sequence data were missing in the combined four protein-coding gene sequence analysis.

All included amino acid sequences passed a chi-square test of compositional

homogeneity at the 5% level, as implemented in TREE-PUZZLE. However, some LSU

rRNA gene sequences (Chrysochromulina, Cryptosporidium Cyanidioschyzon,

Mesostigma, Phaeocystis, Prymnesium) failed compositional homogeneity tests.

Phylogenetic Analysis

Protein-coding gene alignments were analyzed at the amino acid level. Maximum

likelihood (ML) analysis of amino acid sequences was performed using PROML in the

PHYLIP package ver. 3.7 (Felsenstein 2004). A JTT+Γ+I model of protein evolution was

applied with the user-defined Hidden Markov Model (HMM) option for modeling

among-site rate variation. The rates and probabilities for the HMM were estimated from

the neighbor-joining trees using TREE-PUZZLE ver. 5.2 (Schmidt et al. 2002). For each

ML tree search, the input order of sequences was randomized and the process was

repeated 100 times with ‘global rearrangements’. Bootstrap values were obtained from

100 re-samplings, each search with one round of random taxon addition followed by

global rearrangements.

PAUP* 4.0b (Swofford 2002) was utilized for the ML analysis of SSU and LSU

rRNA gene sequences. Modeltest ver. 3.7 (Posada and Crandall 1998) was used to find

the best fitting model of nucleotide evolution and to estimate substitution rates, base

frequencies, Γ distribution parameter (α), and proportion of invariable sites. For each ML



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tree search, the input order of sequences was randomized and the process was repeated

100 times with the tree bisection and reconnection branch-swapping algorithm. Bootstrap

values were obtained from 100 re-samplings, each search with one round of random

taxon addition and the nearest-neighbor interchange branch-swapping algorithm.

Bayesian inference of phylogeny was performed using MrBayes ver. 3.1

(Huelsenbeck and Ronquist 2001). For DNA sequence analyses, the GTR+Γ+I model of

evolution was applied. For protein sequence analyses, the WAG+Γ+I model of evolution

was used. Preliminary Markov Chain Monte Carlo (MCMC) runs with about 10,000

generations of trees were used to find the optimal temperature values, which seemed to

be an important factor in chain mixing (data not shown). At least two independent

MCMC runs were then completed and were compared to assess the reliability of each

run. A total of 1,000,000-2,000,000 generations of trees were selected and evaluated, and

every hundredth tree was sampled for further analysis. The burn-in period was evaluated

using Gnuplot ver. 4.0 (Williams and Kelly 1998).

The approximately unbiased (AU) test was performed to compare three

competing hypotheses related to the phylogenetic position of A. proboscidea relative to

Opisthokonta and Amoebozoa. Tree topologies reflecting these hypotheses were

generated by rearrangement of the ML tree for the dataset (if required). Site likelihoods

for each topology were calculated using TREE-PUZZLE. The AU test was performed

using CONSEL ver. 0.1h (Shimodaira and Hasegawa 2001). The output file from TREE-

PUZZLE was converted to a CONSEL-compatible format using a Python script kindly

provided by Jessica Leigh (Department of Biochemistry and Molecular Biology at

Dalhousie University).



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RESULTS

Evolutionary relationships of Apusomonas proboscidea

The phylogenetic position of Apusomonas was well resolved in the ML analysis

of the combined four protein-coding genes (Fig.1). The clade comprising Apusomonas

and Opisthokonta was recovered, and strongly supported, with an ML bootstrap value of

99%. Amoebozoa was sister to the Apusomonadidae+Opisthokonta clade with 99% ML

bootstrap support.

The same relationship between Apusomonas and Opisthokonta was found in the

ML trees based on individual gene sequence analyses of α-tubulin and β-tubulin with

90% and 41% bootstrap values respectively (see Supplementary Material online). The

specific and strongly supported relationship between Apusomonas and Opisthokonta is

still recovered if diplomonads, Carpediemonas, parabasalids and Andalucia incarcerata

are added to the analysis (These sequences are normally the closest relatives to

Opisthokonta in α-tubulin phylogenies—data not shown). Monophyly of Opisthokonta

was not recovered in the actin analysis, but Apusomonas weakly formed a clade with the

opisthokont Amoebidium (27% ML bootstrap value).

In the analysis of Hsp90 gene sequences, both Amastigomonas and Apusomonas

weakly allied with Stramenopiles (13% ML bootstrap value). In this case, neither an

Opisthokonta-Apusomonadidae-Amoebozoa clade, nor an Opisthokonta-Amoebozoa

clade was found (see Supplementary Material online). When slightly longer-branched

sequences were removed, Apusomonadidae instead formed a weak clade with

Amoebozoa and Opisthokonta (for more information about sequences removed, see

Supplementary Material online). Apusomonadidae were sister to Dictyostelium in the ML



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tree, making Amoebozoa paraphyletic, but this relationship was not significantly

supported (17% ML bootstrap value).

In the combined SSU-LSU rRNA gene phylogeny, Opisthokonta,

Apusomonadidae, and Amoebozoa formed a clade in both the ML and Bayesian analyses

(Fig. 2). Apusomonas branched weakly with the concatenated ‘amoebozoan’ sequence

(44% ML bootstrap value). However, if additional (long-branched) Amoebozoan

sequences were included, Apusomonas formed a very weak clade with Opisthokonta

instead (data not shown). SSU rRNA gene analysis also suggested that Apusomonadidae

were related to the naked lobose amoebozoan Vexillifera, but without significant support.

In the LSU rRNA gene phylogenies, Apusomonas weakly branched within unresolved

clades of biflagellates, not closely related to the included amoebozoan (Mastigamoeba).

Bayesian analysis of the six combined gene sequences indicated that Apusomonas

is closely related to Opisthokonta, with Amoebozoa falling as the sister group to

Apusomonadidae+Opisthokonta (Fig 4). Most deep divergences including these clades

were resolved with Bayesian posterior probabilities of 1. The more conservative ML

bootstrapping approach could not be applied to this mixed amino acid/nucleotide data set.

Our data were used to compare the following three hypotheses. Hypothesis I is

that Apusomonadidae is sister to Opisthokonta (the hypothesis most strongly supported

by the analyses described above). Hypothesis II is that Apusomonadidae and Amoebozoa

are sisters (supported by some of our phylogenies). Hypothesis III is that Opisthokonta

and Amoebozoa are sisters to the exclusion of Apusomonadidae (consistent with a

‘unikont’ clade). In the AU test based on the combined four proteins, hypotheses II and

III were rejected (p=2 X 10-4, 4 X 10-4 respectively) (see Supplementary Material online).



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The AU test based on α-tubulin alone strongly rejected hypotheses II and III (both p=1 X

10-7). However, the analysis of the combined β-tubulin, actin, and Hsp90 gene sequences

did not reject hypotheses II and III (p=0.539, 0.472 respectively). These results suggest

that the strong rejection signals in the combined protein-coding gene analysis (Fig. 1)

mostly originated from the α-tubulin data set. AU tests based on the combined six gene

sequences also rejected hypotheses II and III (p=2 X 10-4, 4 X 10-5 respectively) (see

Supplementary Material online).

Evolutionary relationships of other protist groups

Our combined protein coding gene sequence analyses found moderate to strong

support (88-95%) for a sister relationship between Alveolata and Stramenopiles. Close

affinity of Kathablepharidae to Cryptophyceae was recovered in both SSU and LSU gene

phylogenies (see Supplementary Material). The combined SSU and LSU gene phylogeny

recovered a Kathablepharidae-Cryptophyceae clade with 100% bootstrap support (Fig 2).

Interestingly, none of the protein phylogenies suggested monophyly of

Kathablepharidae+Cryptophyceae, and the position Kathablepharidae was not resolved

with over 50% ML bootstrap support in any of the protein gene phylogenies, although the

six gene analysis placed it in a clade with Cryptophyceae (Fig 4). Two genes—SSU and

Hsp90—supported a sister relationship between Kathablepharidae+Cryptophyceae and

Glaucophyta, although ML bootstrap was moderate, or weak (see Supplementary

Material).



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DISCUSSION

The evolutionary position of Apusomonadidae

With the goal of clarifying the evolutionary position of Apusomonadidae, we

determined five new Apusomonas gene sequences—those for LSU rRNA, α-tubulin, β-

tubulin, actin, and Hsp90, the first four of which are new to Apusomonadidae. Most

individual gene analyses suggested that Apusomonadidae are closely related to

Opisthokonta or Amoebozoa. α-tubulin, β-tubulin, and actin genes separately provided

evidence of Opisthokonta+Apusomonadidae clade, assuming that the eukaryotic root

does not lie between the two groups. Hsp90 genes, when analyzed without including

some excavate sequences, supported an Apusomonadidae plus Amoebozoa clade.

However, the position of Apusomonadidae based on Hsp90 gene sequences was sensitive

to taxon sampling, and Apusomonadidae sometimes appeared as sister to biflagellate

clades, as found by Stechmann and Cavalier-Smith (2003a). Our LSU rRNA gene

phylogenies likewise did not support the affinity of Apusomonadidae to any particular

group, and the position of Apusomonadidae was sensitive to taxon sampling.

The position of Apusomonadidae in SSU rRNA gene phylogenies has been

unstable. In most cases, inferred topologies were poorly supported except for the earlier

study by Cavalier-Smith and Chao (1995), in which a sister relationship between

Apusomonadidae and Opisthokonta was relatively well supported in maximum

parsimony analyses (84%) and least-squares analyses of Jukes-Cantor distances (96%).

However, in subsequent studies, support for the Apusomonadidae-Opisthokonta clade

was weaker (for example Cavalier-Smith and Chao 2003b), or alternative topologies were



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inferred (for examples Berney, Fahrni, Pawlowski 2004; Fig 2 in Cavalier-Smith, Chao,

Oates 2004).

When α-tubulin gene sequences were included, our analyses strongly supported

an Apusomonadidae-Opisthokonta clade, and AU tests rejected the alternative hypotheses

of an Apusomonadidae-Amoebozoa clade or an Opisthokonta-Amoebozoa clade. When

α-tubulin sequences were excluded, an Apusomonadidae-Opisthokonta clade was

recovered in the ML tree (Fig 3), but two alternative hypotheses—an Apusomonadidae-

Amoebozoa clade and an Opisthokonta-Amoebozoa clade (with Apusomonadidae as

their sister group)—could not be rejected. None of the individual or combined gene data

sets analyzed in this study rejected the Apusomonadidae-Opisthokonta clade when AU

tests were applied. Overall, an Apusomonadidae-Opisthokonta clade is the best supported

hypothesis.

Apusomonadidae is not likely to branch within Opisthokonta because members of

Opisthokonta included in our study formed a strong clade (Fig 1–3). In addition,

Steenkamp, Wright, and Baldauf (2005) reported that Apusomonadidae lack an amino

acid insertion in elongation factor 1-α, a synapomorphic character for Opisthokonta.

While Hampl et al. (2005) and Simpson, Inagaki and Roger (2006) noted that

tubulin gene (α-tubulin in particular) phylogenies conflicted with other gene trees with

respect to the positions of some excavates, we did not find any significant conflict

between the α-tubulin tree and any of the other five gene trees with respect to the

position of Apusomonadidae. Determination of the utility of α-tubulin, or any other

genes as phylogenetic markers for analysis of deep eukaryote divergences would require

the comparative analysis of many gene sequences from diverse eukaryotic groups.



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Some components of our study suggested a close relationship between

Amoebozoa and Apusomonadidae. Amoebozoa includes diverse unicellular and

multicellular eukaryotic organisms for which the only morphological commonality is

amoeboid movement (Walochnik, Michel, Aspöck 2004) (the non-amoeboid flagellates

Phalansterium and Multicilia being likely exceptions [Nikolaev et al. 2006]). Several

molecular analyses have suggested monophyly of Amoebozoa (see Fahrni et al. 2003 for

review), however, without robust statistical support, except when taxon sampling was

low (Bapteste et al. 2002). While there are several major subclades in Amoebozoa, so far

a significant amount of genomic data have only been obtained for a few amoebozoan

taxa, which like Entamoeba, are typically long-branched. This is the reason for the

limited taxon sampling for Amoebozoa in our study. Additional sequence data for other

amoebozoan taxa, such as Phalansterium, Tubulinea, and Fabellinea, are needed to

further explore the relatedness of Apusomonadidae to Amoebozoa.

Richards and Cavalier-Smith (2005) suggested that Opisthokonta and Amoebozoa

(‘unikonts’) form a monophyletic group based on five putative synapomorphies

concerning myosin gene types and sequence features. However, this study did not include

several important eukaryotic taxa, such as Rhizaria and Apusomonadidae. Since our

multigene analyses place Apusomonadidae cladistically within the ‘unikont’ clade,

testing for the presence of ‘unikont’-specific myosin genes and indels in

Apusomonadidae would be particularly valuable. Likewise, determination of whether

Apusomonadidae undergo flagellar transformation, and which flagellum is mature would

be useful because flagellar transformation (in which the posterior flagellum is the mature



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one) is proposed to be a key character distinguishing ‘bikonts’ from ‘unikonts’ (Cavalier-

Smith 2003).

Cavalier-Smith and Chao (2003) united apusomonads with the obscure flagellate

Ancyromonas in a more inclusive taxon Apusozoa, based on the presence of a thecae-like

layer on the dorsal surface of Ancyromonas and a weak affinity between the groups in

some SSU rRNA phylogenies. This relationship requires further confirmation, once more

data are available from Ancyromonas.

Implications of an ‘Apusomonadidae-Opisthokonta’ clade

The two most likely relationships of Apusomonadidae suggested in our

study—the Apusomonadidae-Opisthokonta clade and the Apusomonadidae-Amoebozoa

clade—conflict with two existing hypotheses related to deep eukaryotic divergences. The

first of these hypotheses is that Opisthokonta and Amoebozoa shared a common unikont

ancestor (i.e. with a single flagellum and one basal body), while other eukaryotic groups

were ancestrally bikont (having two flagella and two basal bodies) (Cavalier-Smith

2002). The second widely-cited hypothesis is that bikonts—including

Apusomonadidae—are monophyletic, based primarily on the presence or absence of the

DHFR-TS gene fusion (Stechmann and Cavalier-Smith 2002). As explained below,

neither hypothesis is well supported by existing morphological/genomic data and both are

contra-indicated by our results.

Morphological and our molecular phylogenetic data conflict with a hypothesis of

unikont ancestry for Opisthokonta and/or Amoebozoa: Though flagellate cells of

Opisthokonta (those of choanoflagellates, chytridiomycetes, certain Ichthyosporea



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[=Mesomycetozoea], and male gametes of animals) possess a single flagellum, such cells

typically have a non-flagellated second basal body (Barr 1981; Karpov and Leadbeater

1997), consistent with biflagellate ancestry. The ancestral flagellate condition of

Amoebozoa is more ambiguous because flagellate amoebozoans are relatively

uncommon, and evolutionary relationships among the major subclades of Amoebozoa are

poorly understood (Fahrni et al. 2003; Smirnov et al. 2005). On the one hand, flagellate

members of pelobionts, some Protostelia, Phalansterium, and Multicilia possess a single

flagellum and one basal body per kinetid (a unit consisting of one or more flagellar basal

bodies and any associated fibers, roots, and cytoskeleton). On the other hand, those of

Myxogastria and some Protostelia have two basal bodies and usually two flagella (Olive

1975). Spiegel (1981) suggested that the common ancestor of Protostelia likely possessed

two basal bodies, and that some members of Protostelia had lost their second basal body.

Hence, the ‘unikont’ hypothesis is not particularly well supported by the available

morphological data, even before considering the position of apusomonads.

Because the apusomonads have two basal bodies and two flagella, their possible

positioning within the Opisthokont-Amoebozoa clade makes a biflagellate common

ancestor for this clade more parsimonious. Our molecular study therefore further weakens

the hypothesis of unikont ancestry for Opisthokonta and Amoebozoa (Fig 5). In view of

available morphological and molecular evidence, categorizing the minimum

Opisthokonta+Amoebozoa clade as ‘unikonts’ seems unjustified on present data.

Conflict between our molecular phylogenetic results and interpretations of the

DHFR-TS gene fusion data: Philippe et al. (2000) and Stechmann and Cavalier-Smith

(2002) proposed that possession of two separate, monofunctional DHFR and TS (thyA)



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genes was the archaic condition for eukaryotes, since bacteria, when they possess these

two genes, also produce two separate proteins. Based on this premise, Stechmann and

Cavalier-Smith (2002) used the presence or absence of gene fusion between DHFR and

TS (thyA) to help infer the position of the eukaryotic root. Stechmann and Cavalier-Smith

(2002, 2003b) did not find the DHFR-TS fusion gene in Opisthokonta and Amoebozoa,

but noted that studied representatives of Alveolata, Apusomonadidae, Euglenozoa,

Rhizaria, Stramenopiles, and Viridiplantae have bifunctional DHFR-TS fusion genes.

Therefore, these authors suggested that eukaryotes with this ‘derived’ gene fusion form a

monophyletic group (‘bikonts’), within which the eukaryote root cannot lie. However,

this concept is questionable for several reasons.

First, as Embley and Martin (2006) noted, DHFR-TS fusion data are currently

available for relatively few taxonomic groups, and cannot not be used to infer positions

of lineages such as diplomonads and parabasalids that are devoid of these genes. The

assumption that the DHFR-TS gene fusion represents the derived condition in eukaryotes

is another issue. Since multiple cases of replacement and separation of DHFR and TS

genes have been documented, particularly in bacteria (Philip, Creevey, and McInerney

2005), separate positioning of DHFR and TS (thyA) genes may not represent the archaic

condition in eukaryotes. Alternatively, the DHFR-TS fusion gene could be viewed as the

archaic condition in eukaryotes because the DHFR-TS fusion gene is transcribed into a

single mRNA molecule like bacterial DHFR and TS (thyA) genes, which usually occur in

a single operon (although the gene order is reversed). In contrast, DHFR and TS (thyA)

genes are separately transcribed in Opisthokonta and the amoebozoan Hartmannella

(Stechmann and Cavalier-Smith 2003b). These separate DHFR and TS (thyA) genes may



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have been derived from re-fission of the fused gene. While Stechmann and Cavalier-

Smith (2002) suggested that the reversal of the DHFR-TS gene fusion is improbable,

several examples of fusion and re-fission of other genes over evolutionary time have now

been documented in eukaryotes (Arisue, Hasegawa, Hashimoto 2005; Krauss et al. 2006;

Waller, Slamovits, Keeling 2006). Phylogenetic analyses of the DHFR or TS (thyA) gene

sequences also did not positively support a hypothesis of recent common origin of the

fused genes (Lazar, Zhang, Goodman 1993; Schlichtherle, Roos, Van Houten 1996). In

addition, the DHFR and TS genes may have been subjected to multiple lateral gene

transfer (LGT) events. For example, the amoebozoans Dictyostelium (Leduc et al. 2004)

and Physarum have apparently replaced their TS (thyA) genes with non-homologous TS

(thyX) genes. Lastly, there is a lack of strong independent evidence for the ‘bikont’ clade

supposedly identified by DHFR-TS fusion. Stechmann and Cavalier-Smith (2002)

propose that the presence of flagellar transformation is a second synapomorphy for the

bikont clade (see also Cavalier-Smith 2002), however this idea is complicated by the

unambiguous presence of a form of flagellar transformation in the biflagellate ‘unikont’

Physarum (Wright, Moisand, and Mir 1980). Collectively, these considerations suggest

that the proposal that the DHFR-TS gene fusion represents a single derived evolutionary

event within the diversification of extant eukaryotes is questionable.

Our study, which supports an Apusomonadidae+Opisthokonta clade, places a

fusion-bearing taxon within the only non-fusion-bearing clade of eukaryotes. If our

phylogenetic placement of Apusomonadidae is correct, this implies one of two

possibilities: i) The DHFR-TS fusion was laterally transferred at least once (or that the

fusion event occurred more than once), and hence is an unreliable phylogenetic marker,



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or, ii) The DHFR-TS fusion represents an unique evolutionary event, but this took place

before the divergence of extant eukaryotes, and hence is an ancestral character state

(plesiomorphy) for all living eukaryotes. Therefore our study adds substantial additional

doubt as to the validity of the DHFR-TS fusion as a marker for deep eukaryote

diversification, and the monophyly of the group identified by the fusion (‘bikonts’).

Evolutionary relationships of other groups

Our new sequences for representatives of Alveolata, Cryptophyceae,

Glaucophyta, Haptophyta, Stramenopiles, Kathablepharidae, Rhizaria, and Viridiplantae,

allowed us to evaluate additional relationships among major eukaryotic lineages. Our

LSU rRNA gene phylogeny confirmed the previous result of SSU rRNA phylogeny, that

Kathablepharidae and Cryptophyceae are sister taxa (Okamoto and Inouye 2005).

Alveolata and Stramenopiles were sisters in multiple protein gene phylogenies in our

analyses (Fig 1, 3), consistent with previous multi-protein analyses (Baldauf et al. 2000;

Harper, Waanders, Keeling 2005; Simpson, Inagaki, Roger 2006). In the combined SSU

and LSU rRNA phylogeny (Fig 2), however, Alveolata branched weakly with Rhizaria,

which was also observed in some previous SSU rRNA gene analyses (for example Fig 1

and 2 in Cavalier-Smith and Chao 2003a)

None of our analyses suggested the monophyly of “chromalveolates”, including

Alveolata, Cryptophyceae (+Kathablepharidae), Haptophyta, and Stramenopiles

(Cavalier-Smith 1999), particularly with respect to the Kathablepharidae-Cryptophyceae

clade. The “chromalveolate hypothesis”, advocating a single red algal plastid origin for

“chromalveolates” and the monophyly of these groups, is currently hotly debated



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(Falkowski et al. 2004; Grzebyk et al. 2004; Keeling et al. 2004). Because our individual

gene analyses of SSU rRNA and Hsp90 as well as previous Hsp70 phylogeny (Rensing et

al. 1997) suggested, albeit without strong support, close affinity of the Kathablepharidae-

Cryptophyceae clade (or Cryptophyceae) to Glaucophyta, additional genomic data from

Kathablepharidae and Cryptophyceae would be useful to further evaluate their

phylogenetic relationship to other putative “chromalveolate” groups.

Supplementary Material

Supplementary Figures S1–S6 represent ML trees based on analyses of individual

gene sequences. Table S1 shows GenBank accession numbers of newly obtained

sequences in this study. Table S2 shows AU test results. Method S1 includes

supplementary method information.

Acknowledgements

This research was supported by grant MCB-9977903 from the National Science

Foundation, a Davis Summer Research Fellowship (Department of Botany at the

University of Wisconsin-Madison), an Anna Grant Birge Memorial Award (University of

Wisconsin-Madison), and NSERC grant 298366-04 to AGBS. Y. Van de Peer at Ghent

University kindly provided LSU rRNA sequence alignments. The authors also thank B.

Larget (University of Wisconsin-Madison) for access to a computation facility, J. Graham

(University of Wisconsin-Madison) for the Peridinium limbatum culture, L. Wilcox

(University of Wisconsin-Madison) for obtaining samples from aquatic habitats, and J.

Leigh (Dalhousie University) for a Python script.



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TABLE 1. Primers for PCR amplifying and sequencing nuclear-encoded SSU rRNA, LSU rRNA, α-tubulin, β-tubulin, actin, andHsp90 genes. Primer positions are relative to location within Arabidopsis thaliana (LSU rRNA) or Chlamydomonas reinhardtii (allothers).

Primer Other names 5′ end 3′ end Primer sequence Referencenu-SSU Primersnu-SSU-0024-5′ NSF4/21 0004 0024 CTG GTT GAT CCT GCC AGT AGT This studynu-SSU-0033-5′ NSF13/21 0013 0033 CCT GCC AGT AGT CAT AYG CTT This studynu-SSU-0977-5′ NSF963/18 0960 0977 TTR ATC AAG AAC GAA AGT This studynu-SSU-1173-3′ NSR1197/24 1196 1173 CCC GTG TTG AGT CAA ATT AAG CCG This studynu-SSU-1757-3′ NSR1784/21 1777 1757 CAG GTT CAC CTA CGG AAA CCT This studynu-SSU-1768-3′ NSR1795/21 1788 1768 TGA TCC TTC YGC AGG TTC ACC This studynu-LSU Primersnu-LSU-0046-5′ NLF184/23 0024 0046 ACC CGC TGA AYT TAA GCA TAT CA This studynu-LSU-0058-5′ NLF196/23 0036 0058 TAA GCA TAT CAM TAA GCG GAG GA This studynu-LSU-1152-5′ NLF1280/23 1130 1152 TTT GGT AAG CAG AAC TGG CGA TG This studynu-LSU-1262-3′ NLR1431/23 1284 1262 AGT TGT TAC ACA CTC CTT AGC GG This studynu-LSU-2199-5′ NLF2343/24 2176 2199 TGA TTT CTG CCC AGT GCT CTG AAT This studynu-LSU-2383-3′ NLR2571/22 2404 2383 CTC AAC AGG GTC TTC TTT CCC C This studynu-LSU-3100-3′ NLR3287/23 3122 3100 GGA TTC TGR CTT AGA GGC GTT CA This studyα-Tubulin Primersnu-αTUB-0044-5′ TUAF22/23 0022 0044 CAC ATC GGN CAR GCC GGN RTC CA This studynu-αTUB-0083-5′ TUAF58/25 0058 0083 TGC TGG GAG CTN TAC TGC CTN GAG CA This studynu-αTUB-1219-3′ TUAR1248/26 1244 1219 TCC TCC ATN CCY TCN CCN ACR TAC CA This studynu-αTUB-1237-3′ TUAR1268/26 1262 1237 GCY TCR GAR AAY TCN CCY TCC TCC AT This studyβ-Tubulin Primersnu-βTUB-0050-5′ TUBF28/23 0028 0050 GGN CAG TGY GGN AAC CAG ATY GG This studynu-βTUB-0065-5′ funiv 0040 0065 AAY CAR ATY GGY KC/ideoxyI/ AAR TTY TGG GA This studynu-βTUB-1207-3′ buniv 1232 1207 GCY TC/ideoxyI/ GWR AAY TCC AWY TCG TCC AT This studynu-βTUB-1261-3′ TUBR1294/23 1283 1261 GCN TCC TGG TAC TGY TGR TAC TC This studyActin Primersnu-ACTIN-0054-5′ ACTf-13(s) 0037 0054 GAC AAY GGN WCN GGM ATG TG This study



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nu-ACTIN-0056-5′ ACTf-12 0034 0056 TGC GAC AAY GGN TCN GGM ATG GT This studynu-ACTIN-0060-5′ ACTf-13 0037 0060 GAC AAY GGN TCN GGM ATG GTS AAG This studynu-ACTIN-0071-5′ ACTf-17 0049 0071 GGM ATG TGY AAG GCN GGN TTY GC This studynu-ACTIN-0377-5′ ACT120f 0355 0377 GAR AAR ATG ACN CAR ATH ATG TT This studynu-ACTIN-0400-3′ ACT139b 0419 0400 GCY TGD ATN GCN ACR TAC AT This studynu-ACTIN-1108-3′ ACTb-376 1132 1108 AGA AGC AYT T/ideoxyI/C KGT GNA CRA TNG A This studynu-ACTIN-1111-3′ ACTb-377 1133 1111 TAG AAG CAY TTN CKG TGN ACR AT This studyHSP90 Primersnu-HSP90-0041-5′ HspFA 0022 0041 GAR ACN TTY GCN TTY CAR GC This studynu-HSP90-0083-5′ 100XF 0052 0083 CAG CTG ATG TCC CTG ATC ATY AAY ACN TTY TA Simpson, Lukes, Roger

(2002)nu-HSP90-0389-5′ HspFB 0367 0389 CAR TTY GGT GTB GGY TTY TAC TC This studynu-HSP90-0602-5′ HspFC 0581 0602 TSA AGG ACC TSR TCA AGA AGC A This studynu-HSP90-1390-3′ HspRD 1410 1390 CTC NCC RGT GAT GWA GTA GAT This studynu-HSP90-1732-3′ HspRB2 1754 1732 CGY TCC ATR TTN GCN GAC CAN CC This studynu-HSP90-1741-3′ HspRA 1761 1741 CAT GAT NCG YTC CAT RTT NGC This studynu-HSP90-1759-3′ 880XR 1781 1759 TCG CGC AGR GCY TGN GCR TTC AT Simpson, Inagaki,

Roger (2006)nu-HSP90-1809-3′ HspRC 1833 1809 GGG GTT GAT TTC CAT NGT YTT CTT G This studynu-HSP90-1813-3′ 910XR 1835 1813 TCG GGG TTG ATY TCC ATN GTY TT Simpson, Inagaki,

Roger (2006)nu-HSP90-1990-3′ 970XR 2015 1990 TCG AGG GAG AGR CCN ARC TTR ATC AT Simpson, Lukes, Roger

(2002)



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FIGURE LEGENDS

Fig. 1. ML tree (JTT+Γ+I, 8 rate categories) inferred from four nuclear encoded protein

coding gene sequences. The data set included 26 taxa and 1594 amino acid positions. The

root was arbitrarily placed between the Apusomonadidae-Opisthokonta-Amoebozoa

clade and the other taxa. Bayesian analysis (WAG+Γ+I, 8 rate categories) also found a

similar topology. ML bootstrap values and Bayesian posterior probabilities are indicated

at the corresponding nodes. Bootstrap values ≥50% and posterior probabilities ≥0.5 are

shown. Dashes represent bootstrap values <50%. Taxa from which new sequences were

obtained in our study are labeled in boldface.

Fig. 2. ML tree (GTR+Γ+I, 8 rate categories) was inferred from the combined nuclear

encoded SSU rRNA and LSU rRNA gene data set. The data set included 48 taxa and

3287 nucleotide positions—1283 from SSU rRNA and 2004 from LSU rRNA. Statistical

support values are listed in the same way as Fig. 1. The only exception is for the node

leading to Apusomonas. The SSU rRNA gene sequence of Mastigamoeba balamuthi

was substituted with that of Vexillifera armata to reduce the overall branch length of

Amoebozoa. When the SSU rRNA sequence of M. balamuthi was used, Mastigamoeba

branched with the Cryptophyceae+Kathablepharidae clade, while Apusomonas remained

branching close to Opisthokonta.

Fig. 3. ML tree inferred from the same data set used in Fig. 1 without α-tubulin gene

sequences. The same position for Apusomonas (Hypothesis I) was recovered. Statistical

support values for hypothesis II and III are also indicated.



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Fig. 4. Bayesian consensus tree based on six genes. A GTR+Γ+I model (8 rate

categories) of nucleotide evolution was applied to the concatenated SSU and LSU rRNA

gene sequences and a WAG+Γ+I model (8 rate categories) of protein evolution was

applied to the combined four protein-coding genes. Bayesian posterior probabilities are

shown at the corresponding nodes.

Fig. 5. A simplified unrooted tree showing the main conclusion of this study. Regardless

of the position of the eukaryote root, ‘unikonts’ do not form a monophyletic group.



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Apusomonas proboscidea

Caenorhabditis elegans

Suberites/Discodermia

Monosiga brevicollis

Corallochytrium limacisporum

Physarum/Dictyostelium

Bigelowiella natans

Thaumatomonas sp.

Euglena gracilis

Naegleria gruberi

Heterosigma akashiwo

Synura sphagnicola

Phytophthora palmivora

Peridinium/Kryptoperidinium

Perkinsus marinus

Plasmodium sp.

Toxoplasma gondii

Chlamydomonas reinhardtii

Mesostigma viride

Pterosperma cristatum

Cyanophora paradoxa

Goniomonas truncata

Guillardia theta

Chrysochromulina sp. NIES 1333

Isochrysis galbana

Leucocryptos marina

0.05 substitutions/site

77

89

99

99

99

100

100

100

97

77

98

100

100

99

100

88

1

1

1

1

1

1

1

1

1

1

1

1

1

–/.93

.94

.96

1

.71

.98

1

1

.91

–/.96

FIG . 1.

OP

ISTH

OK

ON

TA

APUSOMONADIDAE

RHIZARIA

AMOEBOZOA

EUGLENOZOA

HETEROLOBOSEA

STR

AM

ENO

PIL

ESA

LVEO

LATA

VIRIDIPLANTAE

GLAUCOPHYTA

CRYPTOPHYCEAE

KATHABLEPHARIDAE

HAPTOPHYTA



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Apusomonas proboscideaVexillifera/Mastigamoeba

Blastocladiella emersonii

Pneumocystis cariniiSaccharomyces cerevisiae

Tricholoma matsutakeUmbelopsis ramanniana

Chytriomyces hyalinusOedogoniomyces sp.Nuclearia simplex

Hydra circumcincta

Leucosolenia sp.Mnemiopsis leidyi

Suberites ficusIchthyophonus hoferi

Monosiga brevicollisSalpingoeca infusionum

Aureococcus anophagefferensNannochloropsis salina

Heterosigma akashiwoScytosiphon lomentaria

Tribonema aequaleSynura sphagnicola

Cylindrotheca closteriumHyphochytrium catenoides

Phytophthora sp.Bigelowiella natans

Thaumatomonas sp.Cryptosporidium parvum

Toxoplasma gondiiPerkinsus andrewsi

Prorocentrum micansPhaeocystis antarcticaPrymnesium patelliferum

Bangia atropurpureaCyanidioschyzon merolae

Chlamydomonas sp.Chlorella ellipsoidea

Pterosperma cristatumFunaria hygrometrica

Gnetum gnemonOryza sativa

Mesostigma virideCyanophora paradoxa

Glaucocystis nostochinearumGoniomonas truncata

Guillardia thetaLeucocryptos marina


44/.9

73/1

65/1

100

80

100

100/1

92/1

100

57/.94

65/.97100/1

99

100

98

80/.99

98100

100

95/1

100

98/1

95/158/.93

1

73/.87

1 –/.86

87/175/1

53/1

–/1

66/.99

1

1

1

–/.98

1

1

.85

1

–/1

–/.94

/1

–/.99 100/197/1

1

100/182/1

74/.99–/.97

1

100/1

74/1

FIG. 2.

HAPTOPHYTA

ALVEOLATA

STRAMENOPILES

RHIZARIA

RHODOPHYTA

VIRIDIPLANTAE

CRYPTOPHYCEAE

KATHABLEPHARIDAE

GLAUCOPHYTA

OPISTHOKONTA

APUSOMONADIDAE

AMOEBOZOA



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Caenorhabditis elegans

Suberites/Discodermia


Corallochytrium limacisporum

Physarum/Dictyostelium

Bigelowiella natans

Thaumatomonas sp.

Euglena gracilis

Naegleria gruberi


Synura sphagnicola

Phytophthora palmivora

Peridinium/Kryptoperidinium

Perkinsus marinus

Plasmodium hybrid

Toxoplasma gondii

Chlamydomonas reinhardtii

Mesostigma viride



Isochrysis galbana

Cyanophora paradoxa

Goniomonas truncata

Guillardia theta

Leucocryptos marina


84

100

42

88

99

100

99

91

100

63

54

99

100

98

100

95

54

1

1

1

1

1

1

1

1

1

.99

1

1

.76

.89

.83

1

1

1

1

.96.32

FIG. 3.

40/.35

13/.33

Hyp. I

Hyp. II

Hyp. III

APUSOMONADIDAE

OP

ISTH

OK

ON

TA

AMOEBOZOA

RHIZARIA

EUGLENOZOA

HETEROLOBOSEA

ALV

EOLA

TAST

RA

MEN

OP

ILES

VIRIDIPLANTAE

GLAUCOPHYTA

CRYPTOPHYCEAE

KATHABLEPHARIDAE

HAPTOPHYTA



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Coprinopsis/Cryptococcus/Tricholoma


Discodermia/Suberites

Dictyostelium/Mastigamoeba/Physarum/Vexillifera

Bigelowiella natans

Thaumatomonas sp.


Synura sphagnicola

Cylindrotheca/Thalassiosira

Phytophthora sp.

Kryptoperidinium/Peridinium/Prorocentrum

Perkinsus sp.

Toxoplasma gondii


Chlamydomonas sp.


Mesostigma viride

Cyanophora paradoxa

Goniomonas truncata

Guillardia theta

Leucocryptos marina


1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

.72

1

FIG . 4.

KATHABLEPHARIDAE

HAPTOPHYTA

CRYPTOPHYCEAE

GLAUCOPHYTA

VIRIDIPLANTAEA

LVEO

LATA

STR

AM

ENO

PIL

ES

RHIZARIA

OP

ISTH

OK

ON

TA

APUSOMONADIDAE

AMOEBOZOA



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Apusomonadidae ('bikonts')

Opisthokonta ('unikonts')

Amoebozoa('unikonts')

Other eukaryotes ('bikonts')

FIG 5. by guest on M

arch 10, 2016http://m

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Evolutionary Relationships of Apusomonads Inferred from Taxon-Rich Analyses of 6 Nuclear Encoded...

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