Current Biology, Vol. 12, 1773–1778, October 15, 2002, 2002 Elsevier Science Ltd. All rights reserved.
PII S0960-9822(02)01187-9
The Closest Unicellular Relatives of Animals
B.F. Lang,1,2 C. O’Kelly,1,3 T. Nerad,4 M.W. Gray,1,5
and G. Burger1,2,6
1
The Canadian Institute for Advanced Research
Program in Evolutionary Biology
2
Département de Biochimie
Université de Montréal
Succursale Centre-Ville
Montréal, Québec H3C 3J7
Canada
3
Bigelow Laboratory for Ocean Sciences
P.O. Box 475
180 McKown Point Road
West Boothbay Harbor, Maine 04575
4
American Type Culture Collection
10801 University Boulevard
Manassas, Virginia 20110
5
Department of Biochemistry
and Molecular Biology
Dalhousie University
Halifax, Nova Scotia B3H 4H7
Canada
Summary
Molecular phylogenies support a common ancestry
between animals (Metazoa) and Fungi [1–3], but the
evolutionary descent of the Metazoa from single-celled
eukaryotes (protists) and the nature and taxonomic affiliation of these ancestral protists remain elusive. We addressed this question by sequencing complete mitochondrial genomes from taxonomically diverse protists
to generate a large body of molecular data for phylogenetic analyses. Trees inferred from multiple concatenated mitochondrial protein sequences demonstrate
that animals are specifically affiliated with two morphologically dissimilar unicellular protist taxa: Monosiga brevicollis (Choanoflagellata), a flagellate, and
Amoebidium parasiticum (Ichthyosporea), a funguslike organism. Statistical evaluation of competing evolutionary hypotheses [4] confirms beyond a doubt that
Choanoflagellata and multicellular animals share a
close sister group relationship, originally proposed
more than a century ago on morphological grounds
[5]. For the first time, our trees convincingly resolve
the currently controversial phylogenetic position of
the Ichthyosporea, which the trees place basal to Choanoflagellata and Metazoa but after the divergence of
Fungi. Considering these results, we propose the new
taxonomic group Holozoa, comprising Ichthyosporea,
Choanoflagellata, and Metazoa. Our findings provide
insight into the nature of the animal ancestor and have
broad implications for our understanding of the evolutionary transition from unicellular protists to multicellular animals.
6
Correspondence: [email protected]
Results and Discussion
The evolution of the Metazoa from single-celled protists
is an issue that has intrigued biologists for more than a
century. Early morphological and more recent ultrastructural and molecular studies have converged in supporting the now widely accepted view that animals are
related to Fungi, choanoflagellates, and ichthyosporean
protists. However, controversy persists as to the specific evolutionary relationships among these major
groups. This uncertainty is reflected in the plethora of
published molecular phylogenies that propose virtually
all of the possible alternative tree topologies involving
Choanoflagellata, Fungi, Ichthyosporea, and Metazoa.
For example, a monophyletic Metazoa⫹Choanoflagellata group has been suggested on the basis of small
subunit (SSU) rDNA sequences [1, 6, 7]. Other studies
using the same sequences have allied Choanoflagellata
with the Fungi [8], placed Choanoflagellata prior to the
divergence of animals and Fungi [9], or even placed
them prior to the divergence of green algae and land
plants [10]. Moreover, Ichthyosporea [11], a newly created taxon that was provisionally designated DRIPs (referring to the four initial members, Dermocystidium, rosette agent, Ichthyophonus, and Psorospermium [6])
and later Mesomycetozoa [12], has tentatively been
placed somewhere near [12] (in one case, immediately
before [6]) the animal-fungal divergence. Testifying to
the taxonomic uncertainty surrounding the Ichthyosporea, one class of this phylum, i.e., the Amoebidiales,
had traditionally been classified as trichomycete fungi.
Conflicting scenarios as to the relationship among
Choanoflagellata, Ichthyosporea, and animals were
critically addressed in the course of a recent analysis
using complete SSU and large subunit (LSU) rRNA data,
examined either individually or in combination [13]. This
particular study demonstrates the weakness of tree assessments that are uniquely based on nonparametric
bootstrap values and lends credence to the view that
the precise interpretation of such values is not only difficult [14] but also often leads to overconfidence in the
wrong tree [4]. For example, the monophyly of Choanoflagellata⫹Ichthyosporea is supported by a high bootstrap value (94%) based on the LSU data and by
a marginal bootstrap value (61%) in the combined
LSU⫹SSU data set, whereas the Kishino-Hasegawa
(KH) and Shimodaira-Hasegawa (SH) tests did not recover support for this topology with any combination of
data used [13]. Based upon the highly conflicting results,
the authors come to the conclusion that the available
data may be insufficient to resolve the question of
whether Choanoflagellata, Ichthyosporea, or the two
combined are the closest living relatives of Metazoa.
Basal animal phylogeny has also been investigated via
single nucleus-encoded proteins, but again, the support
for this deep divergence relies solely on the interpretation of often weak and variable bootstrap or quartetpuzzle support values. For instance, trees based on
Hsp70 proteins [15] indicate a closer relationship of
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Figure 1. Maximum Likelihood Tree of Concatenated Proteins Encoded by mtDNA
The sequences of 11 well-conserved proteins
(Cox1,2,3, Cob, Atp6,9, and Nad1,3,4,4L,5)
were concatenated. A ⌫ distribution model
of site variation was used (see Experimental
Procedures). Percent bootstrap support for
PROML (300 samples) is shown above each
branch, and that for PUZZLEBOOT/BIONJ
(1000 samples) is shown below each branch.
The scale bar denotes genetic distance.
Taxon designations are as follows (GenBank
Accession numbers within parentheses):
Magnetospirillum
magnetotacticum
(NC_002725);
Rickettsia
prowazekii
(NC_000963); Chrysodidymus synuroideus
(NC_002174);
Phytophthora
infestans
(NC_002387); Hyaloraphidium curvatum
(NC_003048);
Spizellomyces
punctatus
(NC_003052);
Schizophyllum
commune
(NC_003049);
Podospora
anserina
(NC_001329); Rhizopus stolonifer (unpublished; see Supplementary Material); Allomyces macrogynus (NC_001715); Sarcophyton
glaucum
(AF064823,
AF063191);
Metridium senile (NC_000933); Homo sapiens
(J01415); Monosiga brevicollis (this publication); Prototheca wickerhamii (NC_001613);
Marchantia polymorpha (NC_001660); Amoebidium parasiticum (this publication); Porphyra purpurea (NC_002007); and Chondrus
crispus (NC_001677).
Choanoflagellata (represented by the taxon Monosiga
ovata) to animals than to Fungi. However, the bootstrap
value for this relationship is low (58%) and, as the authors state [15], “rigorous statistical tests such as the
Kishino-Hasegawa test were also carried out but were
unable to provide statistical support for any of the alternative trees.” Another recent phylogenetic study used
elongation factor 2 (EF-2), ␣- and ␤-tubulin, and actin
proteins [16]. In all four trees, bootstrap and quartetpuzzle indices are only shown at selected branches and
are weak in support of either the fungal-animal divergence or the branching order within the animals, thus
calling into question the overall topology of these trees.
Finally, in this study among-site rate heterogeneity was
not taken into consideration, and no statistical tree selection tests were performed.
We posit that one important reason for the controversy about early animal evolution is that the available
sequence data have been insufficient to yield unambiguous resolution of the taxa in question. To provide a
suitable data set, we sequenced complete mitochondrial genomes from diverse protist phyla [17], including
the choanoflagellate Monosiga brevicollis and the ichthyosporean Amoebidium parasiticum, and thereby
generated the first mitochondrial gene sequences for
Choanoflagellata and Ichthyosporea. With this new data
set, we addressed two specific questions: which of
these present-day protist groups is on the lineage leading specifically to Metazoa, and which one is the closest
living relative of animals? Phylogenetic analyses of
these new data are reported here, with the gene content
and genome architecture of A. parasiticum and M. brevicollis mtDNAs being presented elsewhere.
For phylogenetic analyses, we used our own generated mitochondrial protein sequences, including data
reported here from M. brevicollis and A. parasiticum, as
well as sequences determined by others and retrieved
from public data repositories. The analyses included
about 3000 aligned amino acid positions from 11 wellconserved proteins whose sequences were concatenated (see Experimental Procedures section for details).
Figure 1 depicts the tree obtained with the maximum
likelihood (ML) method implemented in PROML [18],
with site heterogeneity modeled by the discrete ⌫ distribution. Because the ML method is computationally
highly demanding, the number of taxa was limited to 20.
Taxon selection was based on two criteria; we excluded
taxa that either completely lack mitochondrial nad genes
or that display highly accelerated rates of evolution of
mitochondrial proteins (see Experimental Procedures
for details). Notably, within the Metazoa, essentially all
taxa evolve quickly, with the exception of the sea anemone Metridium [19] and the leather coral Sarcophyton
[20]. Therefore, only one metazoan (i.e., human) sequence having a relatively long branch length has been
included in the analyses. The same data set was also
analyzed with an ML distance method (TREE-PUZZLE
and BIONJ [21, 22]), PUZZLEBOOT [23] for bootstrapping, and the same model of site heterogeneity, which
yielded a tree topology identical to that of the ML reconstruction.
Figure 2 shows a tree inferred only with the distance
method. Here, many more species could be included
because distance methods are computationally less demanding than ML analyses. Inclusion of additional animals, Fungi, and plants, and the addition of available
The Closest Unicellular Relatives of Animals
1775
Figure 2. Distance Method Tree of Concatenated Proteins Encoded by mtDNA
The sequences of 11 well-conserved proteins (Cox1,2,3, Cob, Atp6,9, and Nad1,3,4,4L,5) were concatenated. A ⌫ distribution model of site
variation was used (see Experimental Procedures). The number at each branch represents bootstrap support (percent) for PUZZLEBOOT
(1000 samples). Taxon designations, in addition to those specified in Figure 1, are as follows: Sinorhizobium meliloti (NC_003047); Lumbricus
terrestris (NC_001673); Drosophila yakuba (X03240); Branchiostoma lanceolatum (MTY16474); Mus musculus (J01420); Rhizophydium sp.
(NC_003053); Aspergillus nidulans (L19866, X00790, V00650; X15441, X15011, X06960, X06961, AH001255, M35967, J01387, J01388, and
J01389); Pichia canadensis (NC_001762); Yarrowia lipolytica (NC_002659); Acanthamoeba castellanii (NC_001637); Dictyostelium discoideum
(NC_000895); Reclinomonas americana (AF007261); Malawimonas jakobiformis (NC_002553); Mesostigma viride (AF353999); Arabidopsis
thaliana (NC_001284); and Nephroselmis olivata (AF110138).
data from jakobids, did not change the relevant part of
the tree topology or the support in the Choanoflagellata⫹Ichthyosporea⫹Metazoa clade, compared to Figure 1. We observed, however, that support values for
the monophyly of red algae and green algae⫹land plants
decreased as a consequence of the inclusion of jakobids, an issue that will be addressed in more detail
elsewhere. It should be noted that trees were also constructed with individual, well-conserved proteins (Cob,
Cox1) and with three combinations of several proteins
(Cob,Atp6,9; Cox1,2,3; Nad1,3,4,4L,5). The phylogenetic
position of A. parasiticum and M. brevicollis was identical to the one shown in Figures 1 and 2, with four of
these five data sets. However, the resulting trees did
not yield significant support in favor of or against the
topology that was obtained with the full set of concatenated proteins.
When we used the concatenated data sets, both ML
and distance approaches yielded identical tree topologies with branches supported by robust (⬎90%) bootstrap indices. To assess the level of confidence in tree
selection, we performed statistical tests with the soft-
ware programs CONSEL and PAML [24, 25], which provide the least biased and most rigorous tests available
to date [4, 14]. We tested the significance of each set
of competing tree topologies that included Choanoflagellata, Ichthyosporea, Metazoa, and Fungi. The results
of the standard AU test, the weighted KH test (WKH),
weighted SH test (WSH) [4], and bootstrap probability
[18] derived from the data set used in Figure 1 are compiled in Table 1. The standard AU and WKH tests confirm
the topology shown in Figure 1, i.e., they reject all alternative scenarios (at a significance level of 0.05), namely
the Choanoflagellata⫹Ichthyosporea, Ichthyosporea⫹
Metazoa, Ichthyosporea⫹Fungi, and Choanoflagellata⫹
Fungi sister relationships. It should be noted that for the
tree including H. sapiens, the WSH test does not reject
(at the given significance level) the hypothesis that Ichthyosporea are basal to Fungi and Metazoa (Table 1,
topology #3). However, this topology is unambiguously
rejected when H. sapiens is excluded from the data set
(see Supplementary Material). We attribute this difference to the fact that H. sapiens displays the longest
branch, and long branches are notoriously difficult to
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Table 1. Likelihood Tests of Alternative Tree Topologies
Tree Topology
Ta
AUb
BPc
WKHd
WSHe
#1 Best tree (Figure 1)
#2 M.b. basal to Metazoa/Fungi
#3 A.p. prior to Metazoa/Fungi
#4 M.b. and A.p. member of Fungi
#5 A.p. and M.b. sister taxa
#6 A.p. together with Fungi
#7 M.b. member of Fungi
#8 A.p. and M.b. basal to Metridium/Sarcophyton
#9 M.b. basal to Metridium/Sarcophyton
#10 A.p. sister to Metazoa; M.b. basal to A.p.
⫺30.6
30.6
32.8
33.0
37.2
40.4
57.2
140.2
144.3
151.5
0.997
0.003
0.017
0.004
0.001
0.007
0.001
0.001
0.000
0.000
0.980
0.003
0.013
0.002
0.000
0.002
0.000
0.000
0.000
0.005
0.980
0.006
0.020
0.005
0.001
0.004
0.001
0.000
0.000
0.000
1.000
0.020
0.083f
0.017
0.005
0.018
0.002
0.000
0.000
0.000
The program CONSEL [24] was employed, and the same factor for the discrete ⌫ distribution was used as in Figures 1 and 2.
a
Log likelihood difference.
b
Standard approximately unbiased test.
c
Bootstrap probability.
d
Weighted Kishino-Hasegawa test.
e
Weighted Shimodaira-Hasegawa test.
f
This value decreases to 0.032 when the long-branching taxon H. sapiens is removed from the data set.
place with confidence in phylogenetic analyses [26]. We
also emphasize that the developers of the WSH test
regard this test as biased and excessively critical in
assessing the significance of likelihood differences
among competing tree topologies, and they recommend
the AU test for general tree testing [4].
The results shown in Figures 1 and 2 clearly identify
M. brevicollis, and by implication the Choanoflagellata,
as a sister taxon to the Metazoa. This result confirms
the hypothesis that sponges and all other animals
evolved from a choanoflagellate-like ancestor, a proposal made as early as 1866 on the basis of the remarkable morphological similarities between feeding cells
(choanocytes) of sponges and choanoflagellate protists
[5]. The second important conclusion of our results is
that A. parasiticum, and by implication the Ichthyosporea [27, 28], emerged prior to animals and choanoflagellates and clearly after the divergence of the fungi.
Thus, Ichthyosporea, Choanoflagellata, and Metazoa together form a higher-order taxon that we term the Holozoa.
Evolutionary Implications
With the data and analyses presented here, we are now
able to provide unambiguous and compelling evidence
that Choanoflagellata, Ichthyosporea, and Metazoa
constitute a monophyletic assemblage, the Holozoa, to
the exclusion of Fungi and other eukaryotic groups.
Within the Holozoa, Ichthyosporea diverge basally,
whereas Choanoflagellata represent the sister taxon to
animals. Because Choanoflagellata and Ichthyosporea
are both unicellular protist groups, the specific ancestors of animals were most likely unicellular organisms
as well. These postulated single-celled ancestors of
Metazoa must have given rise to multicellular protoanimals, from which the major extant metazoan lineages
(Bilateria, Cnidaria, Ctenophora) subsequently emerged.
Including the findings presented here, we now recognize
a total of five major eukaryotic clades that encompass
both unicellular and multicellular members. These are
the Fungi, Streptophyta (charophyte algae ⫹ land
plants), Rhodophyta (red algae), Phaeophyta (brown al-
gae), and the Holozoa defined here. Hence, multicellular
groups have a phylogenetically dispersed distribution.
This fact, together with the existence of disparate tissue
types, developmental strategies, and cell-cell communication mechanisms provides increasing evidence that
multicellularity is a trait that has emerged independently,
on several occasions, during eukaryotic evolution.
Our results testify to the considerable potential of
mitochondrial genomics as applied to protistan eukaryotes. This approach not only reveals novel types of mitochondrial genome structure and gene expression (for a
review, see [29]), but it also generates large data sets
that are particularly well suited to resolving the phylogenetic relationships of deeply diverging eukaryotic lineages; such relationships cannot be discerned by single-gene analyses.
Experimental Procedures
Strains and Cultivation
M. brevicollis (ATCC 50154) was obtained from the American Type
Culture Collection. The organism was grown in batch cultures at
approximately 25⬚C on sterile natural seawater and fed with live
bacteria (Enterobacter aerogenes ATCC 13048). A. parasiticum JAP7-2 was obtained from R.W. Lichtwardt (Department of Botany,
University of Kansas, Lawrence, KS) and cultured in liquid medium
(1% yeast extract, 3% glycerol) with shaking.
DNA Extraction, Cloning, and Sequencing
Cells of M. brevicollis and A. parasiticum were suspended in sorbitol
buffer (0.6 M sorbitol, 5 mM EDTA, 50 mM Tris [pH 7.4]), broken
mechanically by being shaken with glass beads, and subsequently
lysed in the presence of 1% SDS and 100 ␮g/ml proteinase K.
SDS was eliminated by NaCl precipitation. Total nucleic acids were
fractionated by CsCl/Hoechst 33258 dye isopycnic centrifugation,
whereby mitochondrial DNA forms the uppermost (A⫹T-rich) band,
as verified by hybridization of all fractions with a probe including
the cox1 gene. The upper band was recentrifuged to achieve further
purification. Random clone libraries were constructed by nebulization of the purified mtDNA (into fragment sizes of 1–3 kbp) and
cloning into pBluescript (Stratagene). The corresponding protocol
is available [17]. Clones were sequenced by a combination of automated Li-Cor and manual methods.
Sequence Analysis
Sequence readings were assembled and proofread with the GAP
software suite [30]. The FASTA program [31] was employed for
The Closest Unicellular Relatives of Animals
1777
searches in local databases, and the BLAST network service [32]
was employed for similarity searches in GenBank at the National
Center for Biotechnology Information. Custom-made batch utilities
used for submitting queries and browsing the results are available [17].
Phylogenetic Analyses
For phylogenetic analyses, we used a total of 2969 amino acid
positions from 11 concatenated, well-conserved protein sequences
(Cox1,2,3, Cob, Atp6,9, and Nad1,3,4,4L,5) that are encoded in
mtDNAs of most eukaryotes. Exceptions are Atp9, which is nucleusencoded in Metazoa and in Podospora anserina, and Nad1, 2, and
4, for which complete genes in A. parasiticum have not been identified so far. Multiple sequence alignment was performed with CLUSTAL W [33], and only amino acid positions that could be aligned
without ambiguity were used in the analysis (the data used for the
phylogenetic analysis will be made available, on request). Phylogenetic inferences employed either the ML method as implemented
in PROML [18] or a distance approach. For the calculation, we
used the distance table TREE-PUZZLE [21], which allows a Jin/
Nei correction for unequal rates of change at different amino acid
positions, and we used BIONJ [22] for tree inference. Taxon sampling was given particular consideration. In both distance and ML
analyses, we excluded taxa that either completely lack mitochondrial nad genes (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Plasmodium falciparum) or have highly accelerated
rates of mitochondrial evolution (e.g., ciliates, trypanosomatids, and
green algae of the Chlamydomonas group as well as Pedinomonas
minor). In ML analyses, the number of taxa had to be restricted to
20 per analysis; this was necessary because this method is computationally very demanding, especially when one uses evolutionary
models that permit rate change at all amino acid positions of the
protein sequences and applies subsequent bootstrap analyses. The
20 species used to generate the tree shown in Figure 1 were chosen
to include (i) all fungal and animal species with moderate branch
length; (ii) one representative of higher animals with a long branch
length (human); (iii) two to three representatives each of plants and
green algae, red algae, and stramenopiles; and (iv) a bacterial outgroup with the two ␣-proteobacteria that are most closely related
to mitochondria (Rickettsia and Magnetospirillum). In the distancebased phylogenetic analysis shown in Figure 2, we included additional data from ␣-proteobacteria and all mitochondrial data that
satisfy the requirements specified above, except that the number
of metazoan taxa, which exceeds by far that from all other phyla,
was reduced to five. Bootstrap analysis was applied to both PROML
and TREE-PUZZLE/BIONJ trees. For likelihood tests, P values were
calculated by the CONSEL software [24], and log-likelihood values
were calculated by the PAML package [25]. A ⌫ distribution model
of site variation with an ␣ factor of 0.7, eight categories, and the
JTT matrix were used [34].
Supplementary Material
The ML tree excluding humans, a table showing the corresponding
results of likelihood tests, and the unpublished protein sequences
of Rhizopus stolonifer used in these analyses are available at http://
images.cellpress.com/supmat/supmatin.htm
Acknowledgments
We thank R.W. Lichtwardt (Department of Botany, University of
Kansas, Lawrence, KS) for supplying an axenic culture of A. parasiticum, L. Forget and I. Plante for clone library construction, and Z.
Wang, Y. Zhu, and S. Cagna for DNA sequencing. We also thank the
anonymous referees of the manuscript for constructive suggestions.
This project was supported by a grant from the Canadian Institutes
for Health Research and equipment grants from Sun Microsystems
(Palo Alto, CA) and Li-Cor (Lincoln, NE). Salary and interaction support from the Canadian Institute for Advanced Research to G.B.,
M.W.G., C.J.O., and B.F.L. is gratefully acknowledged.
Received: July 1, 2002
Revised: August 19, 2002
Accepted: August 19, 2002
Published: October 15, 2002
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Accession Numbers
The GenBank accession numbers for the sequences reported in
this paper are AF538042-AF538052 (A. parasiticum mtDNA) and
AF538053 (M. brevicollis mtDNA).