Phylogenetic Analysis of the Hsp70 Sequences Reveals the Monophyly of
Metazoa and Specific Phylogenetic Relationships Between
Animals and Fungi
C. Borchiellini, N. Boury-Esnault, J. Vacelet, and Y. Le Parco
Centre d’Océanologie de Marseille, Station Marine d’Endoume, Université de la Méditerranée, Marseille, France
To understand the early evolution of the Metazoa, it is necessary to determine the correct phylogenetic status of
diploblastic animals. Despite cladistic studies of morphological characters and recent molecular phylogenetic studies,
it remains uncertain whether diploblasts are monophyletic or paraphyletic, and how the phyla of diploblasts are
phylogenetically related. The heat shock protein 70 (Hsp70) sequences, because of their ubiquity and high degree
of conservation, could provide a useful model for phylogenetic analysis. We have sequenced almost the entire
nucleic acid sequence of cytoplasmic Hsp70 from eight diploblastic species. Our data support the monophyly of
diploblastic animals. However, the phylogenetic relationships of the diploblast groups were not significantly resolved. Our phylogenetic trees also support the monophyly of Metazoa with high bootstrap values, indicating that
animals form an extremely robust clade.
Introduction
On the basis of long and thorough studies of comparative morphology, animals have been divided into
three major taxonomic units, namely, triploblasts, diploblasts, and animals with extremely loose tissue differentiation: the Parazoa (sponges). At any rate, in all
schemes, sponges are always placed at the base of the
tree as the earliest emerging line because of their ‘‘simplicity’’ and some deep differences from the rest of the
metazoa. The discussion centers on whether the possession of collagen and typical metazoan spermatozoa is
sufficient to unite them with the rest of the animals.
Thus, sponges have been classified within the diploblasts.
The phylogenetic relationships between early animal phyla such as the Porifera, Ctenophora, and Cnidaria are of central importance for understanding the
evolutionary pathway the Metazoa followed from unicellular organisms. An unequivocal phylogeny of diploblastic phyla has not emerged from morphological,
biochemical, and physiological evidence because of the
tremendous diversity of these data and the scarcity of
the fossil record in the Precambrian. However, it is essential to determine the early branching order of animal
phyla in order to study the evolution of various molecular, cellular, and developmental characters. Despite numerous phylogenetic analyses using methods of molecular phylogeny and rRNA 18S or 28S as a phylogenetic
index (Field et al. 1988; Ghiselin 1989; Patterson 1989,
1990; Walker, Bode, and Steele 1989; Hendriks et al.
1990; Lake 1990; Christen et al. 1991; Adoutte and Philippe 1993; Wainright et al. 1993; West and Powers
1993; Cavalier-Smith, Allsopp, and Chao 1994a, 1994b;
Cavalier-Smith et al. 1996), three major issues in relation to the phylogenetic status of diploblasts remain
poorly resolved: whether all the metazoans including diKey words: heat shock protein, phylogeny, diploblasts, sponges.
Address for correspondence and reprints: C. Borchiellini, Centre
d’Océanologie de Marseille, Station Marine d’Endoume, Université de
la Méditerranée, UMR-CNRS 6540, rue de la Batterie des Lions,
13007 Marseille, France. E-mail: [email protected].
Mol. Biol. Evol. 15(6):647–655. 1998
q 1998 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
ploblasts and triploblasts are monophyletic, whether the
diploblasts are monophyletic or paraphyletic, and how
the diploblast phyla are related.
It has been suggested that ribosomal gene sequences are unable to resolve patterns of relationships that
span more than 500 Myr (Rodrigo et al. 1994). One
plausible reason for the apparent inability of ribosomal
sequences to provide a strong and accurate phylogenetic
signal over this time period is that any phylogenetic information present in the data has been lost (Rodrigo et
al. 1994).
The use of molecular sequence data to deduce deep
phylogenetic relationships relies upon informational
macromolecules which are ubiquitous and whose primary structure as well as function is highly conserved
during evolution (Woese 1987; Woese, Kandler, and
Wheelis 1990; Müller 1995). The Hsp70 family of proteins constitutes one of the most conserved proteins
known to date that is found in all species. In the past
8–10 years, because of the perceived importance of
Hsp70 in cell structure and function, the cDNA/genes
for Hsp70 have been sequenced from a large number of
prokaryotic and eukaryotic species. In eukaryotic cells,
several distinct Hsp70 homologs, many of which are
localized in different intracellular compartments, have
been identified.
Members of the Hsp70 family carry out a highly
conserved molecular chaperone function in the intracellular transport of proteins and in protecting the organisms from thermal or other stress-induced damage
(Lindquist and Craig 1988; Morimoto, Tissieres, and
Georgopoulos 1990; Gething and Sambrook 1992).
These studies revealed that the primary structure of
Hsp70 is highly conserved during evolution (Lindquist
and Craig 1988; Gupta and Singh 1992). The high degree of sequence conservation of Hsp70, in conjunction
with a large size and an ancient conserved function,
makes it a particularly useful system for investigating
deep phylogenetic relationships (Gupta and Golding
1993; Koziol et al. 1998). The eukaryotic Hsp70s comprise four distinct clusters corresponding to the intracellular localization of the proteins (Boorstein, Ziegel647
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Borchiellini et al.
Table 1
Species Used in this Study
Species
Porifera
Class Demospongiae
Tetractinellida . . . . .
Chondrosida . . . . . .
Poecilosclerida . . . .
Haplosclerida . . . . .
Geodia cydoniuma
Chondrosia reniformis
Asbestopluma hypogea
Petrosia ficiformis
Author
Collecting Sites
(Jameson, 1811)
Nardo, 1847
Vacelet and Boury-Esnault, 1996
(Poiret, 1789)
Adriatic Sea, Rovinj
Mediterranean Sea, Marseille, France
Mediterranean Sea, La Ciotat, France
Mediterranean Sea, Marseille, France
Class Calcarea
Calcaronea . . . . . . . Petrobiona massiliana
Vacelet and Lévi, 1958
Mediterranean Sea, Marseille, France
Class Hexactinellida
Hexasteorphora . . . . Rhabdocalyptus dawsoni
(Lambe, 1892)
Pacific Ocean, Barkley Sound, Canada
Cnidaria
Class Anthozoa
Octocorallia. . . . . . . Funiculina quadrangularis
Eunicella cavolini
(Pallas, 1766)
(Koch, 1887)
Mediterranean Sea, Marseille, France
Mediterranean Sea, Cassis, France
Ctenophora
Class Nuda . . . . . . . . . Beroe ovata
Eschscholtz
Mediterranean Sea, Villefranche/mer, France
hoffer, and Craig 1994). Members of individual eukaryotic subgroups are more similar to each other. Members
of cytoplasmic Hsp70 cluster are particularly similar,
sharing at least 71% identity (Boorstein, Ziegelhoffer,
and Craig 1994). However, highly conserved residues
do occur near the C- and N-termini within subclasses of
Hsp70s.
In this study, to assess some of the relationships
outlined above, we used the polymerase chain reaction
(PCR) to amplify, clone, and sequence almost the entire
nucleic acid sequence of the cytoplasmic Hsp70 orthologous gene from eight new diploblastic species. In order
to clone only the orthologous cytoplasmic Hsp70 gene
in each species, we identified highly conserved regions
at the start and at the end of the translated regions of
the cytoplasmic Hsp70 orthologs that enabled us to synthetize specific degenerated primers (see Materials and
Methods).
We compared the nucleotide sequences at the first
and second codon positions. Phylogenetic trees were inferred by neighbor-joining and maximum parsimony,
and the validity of the results was ascertained by bootstrapping.
Results presented in this paper confirm that the Metazoa are monophyletic and emerge in two distinct
branches leading, respectively, to diploblasts and triploblasts. However, this early dichotomy is poorly supported by bootstrap analysis as well as by the phylogenetic relationships within the diploblast subtree.
molecular analysis from the interior of the specimen to
avoid possible subsurface unicellular algal contamination. Tissues were fixed in 80% ethanol and stored at
2208C.
Before DNA genomic extraction, tissue samples
were dehydrated and frozen. Small pieces of frozen samples were ground to a powder in a precooled mortar with
liquid nitrogen. Powder was placed in 500 ml lysis buffer (10 mM Tris-HCl [pH 8], 0.1 M EDTA [pH 8], 20
mg/ml RNAse DNAse free, 0.5% SDS) and incubated
for 1 h at 378C. The mixture was digested by addition
of Proteinase K (100 mg/ml) for 3 h at 508C. After digestion, the aqueous lysate was extracted with watersaturated ultrapure phenol, followed by a single chloroform extraction of the aqueous phase. Genomic DNA
was recovered by standard precipitation procedures with
0.1 volume of 3 M ammonium acetate (pH 7) and 2.5
volumes of absolute ethanol. Genomic DNA was finally
resuspended in sterile water at 1 mg/ml after measurement at 260/280 nm.
a
This Hsp70 sequence was obtained from GenBank.
Materials and Methods
Sample Collection and Genomic DNA Extraction
Determination of the Primers
We carried out a general alignment of all Hsp70
sequences available in GenBank and used this alignment
to deduce a number of signature sequences distinctive
of the Hsp70 family in order to distinguish specific
regions for the different Hsp70 paralogous genes. We
identified highly conserved regions at the C- and Nterminii sequences for the cytoplasmic Hsp70 orthologs
that enabled us to synthetize degenerated primers in order to clone the orthologous gene in each diploblastic
species.
Sponges and cnidarians were collected by scuba
diving and ctenophores were collected with plankton
nets from the Mediterranean Sea and the Pacific Ocean.
The list of species used in this study and the collecting
sites are shown in table 1. After collection, animals were
cleaned of epibionts, and a small amount were taken for
Isolation of Cytoplasmic Hsp70 Genes
Hsp70 genes were isolated using PCR. The first
PCR was performed using specific degenerated primers
of 59 (59-GG(CTG)AC(ACGT)AC(CGT)TA(CT)TC(ACGT)TG(TC)GT-39) (GTTYSCV) and 39 (59TAGTC(AT)AC(CT)TC(CT)TC(GT)AT(GA)GT-39)
Monophyly of Metazoa Inferred by Hsp70 Sequences
(PTIEEVD) regions of the cytoplasmic Hsp70 gene. Before amplification, the reaction mixture was denatured
at 948C for 5 min. Amplification of genomic DNA was
undertaken with a DNA thermal cycler (Perkin Elmer
Celtus) in a final volume of 50 ml in the presence of 10
ng of genomic DNA, 5 ml of 10 3 Taq DNA polymerase buffer, 8 ml of 1.25 mM dNTP mix (Pharmacia), 2.5
ml of each primer (20 mM) and 1.25 U of Taq DNA
polymerase (Promega). Samples were amplified for 30
cycles under the following regime: denaturation at 948C
for 1 min, primer annealing for 1 min at 528C, and extension for 3 min at 728C. PCR amplification products
were extracted from agarose gel using the Giaex II gel
extraction kit (Qiagen) according to the manufacturer’s
instructions. These products of first amplification were
reamplified using internal degenerated primers 59AT(TCA)AT(TC)GC(TC)AA(TC)GA(ATGC)CA(AG)GG-39 (IIRNEQ) and 59-CC(CT)TT(AG)TC(AG)TT(ACGT)GT(TAG)AT(TAG)GT-39 (TITNDK). For this
second amplification, samples were amplified under the
following conditions: denaturation at 948C for 1 min,
primer annealing for 1 min at 608C, and extension for
2 min at 728C. The 1,500-base region of genomic Hsp70
gene was cloned into pTZBlue T-Vector (Tebu) and sequenced by dideoxy-nucleotide chain termination (Sanger, Nicklen, and Coulson 1977).
Sequence Comparison
Sequences were aligned using the GeneWork program and manually for correction of any obvious misalignments. Trees were derived from molecular data using the distance matrix neighbor-joining method (Saitou
and Nei 1987) and the maximum-parsimony algorithms
in the PHYLIP package (Felsenstein 1992) in the PHYLO-WIN package (Galtier, Gouy, and Gautier 1996).
Distance matrices were calculated using the observeddivergence method. The validity of the results was ascertained with bootstrapping (Felsenstein 1992).
Nucleotide Sequence Accession Numbers
The Hsp70 gene sequences corresponding to the
diploblastic species identified were deposited in the
GenBank database. The accession numbers are: Beroe
ovata (AF026512), Asbestopluma hypogea (AF026513),
Petrosia ficiformis (AF026514), Rhabdocalyptus dawsoni (AF026515), Funiculina quadrangularis (AF026516),
Chondrosia reniformis (AF026517), Eunicella cavolini
(AF026518), and Petrobiona massiliana (AF026520).
The reported Hsp70 sequences for the following
species were also used in this study: (1) metazoans—
Bruggia malavi (M68933), Caenorhabditis elegans
(M18540), Ceratitis capitata (U20256), Drosophila
melanogaster (J01103), Echinococcus granulosus
(U26448), Mesocestoides corti (U70213), and Geodia
cydonium (X94985); (2) fungi—Saccharomyces cerevisiae (Z35836) and Pichia angusta (Z29379); (3)
plants—Arabidopsis thaliana (X77199) and Lycopersicon esculentum (L41253); (4) Alveolata—Euplotes eurystomus (L15292) and Oxytricha nova (U37280).
649
Results
Relationships Between Diploblastic Animals
We obtained partial cytoplasmic Hsp70 sequences
from eight diploblastic metazoa (table 1). The deduced
amino acid sequences were aligned with translations of
several previously published cytoplasmic Hsp70 genes
(fig. 1). A consensus neighbor-joining tree based on the
nucleotide sequences of diploblastic animals, obtained
after 500 bootstrap replicates, is presented in figure 2.
The tree was rooted using the Hsp70 ortholog from two
representatives of the Alveolata. In this tree, the Cnidaria diverge first, followed by the Ctenophora, which
form the sister group to the Porifera lineage. The sponges form a relatively well supported monophyletic clade
(88% of the bootstrap replicates) in which the Hexactinellida emerge at the base as the sister group to the
Demospongiae-Calcarea cluster. The relationship between Demospongiae and Calcarea classes is supported
by 80% of the bootstrap replicates. The evolutionary
relationship between Hsp70 sequences was also examined by parsimony analysis (fig. 3). The results of a
bootstrap (maximum-parsimony) analysis for the robustness of the data are shown as percentages indicated
above each branch. The branching pattern of species in
the parsimony tree is different from that observed in the
neighbor-joining tree. Figure 3 groups the Hexactinellida with the Ctenophora rather than with the other Porifera. However, the bootstrap support for this grouping
is weak (51%). The low bootstrap score in this case is
due to the uncertain branching position of the Hexactinellida. The representative of the Hexactinellida class
often branched with the Ctenophora (see fig. 3), while
in the other bootstrap sets (data not shown), Hexactinellida branched with other Porifera species. Thus, the
monophyly of sponges is more open to question.
Monophyly of Metazoa and Relationships Between
Animals, Plants, and Fungi
In order to study the phylogenetic relationship between triploblasts and diploblasts and, more broadly, the
branching order between animals, plants, and fungi, our
sequences of ‘‘lower’’ metazoan taxa were aligned with
existing Hsp70 orthologous sequences from a range of
higher plants and fungi and several sequences of triploblastic animals extracted from GenBank (see Material
and Methods).
Phylogenetic trees were constructed by both neighbor-joining (fig. 4) and parsimony (data not shown)
methods. The two methods yielded identical branching
orders. Figure 4 confirms the classical distinction between diploblastic and triploblastic metazoans, but the
diversification of diploblastic animals into sponges, cnidarians, and ctenophores occurred in our tree after the
split between diploblasts and triploblasts. However,
bootstrap analysis provided only weak support for this
early dichotomy of animals. The branching pattern in
the parsimony tree, identical to that observed in the
neighbor-joining tree, confirms the early emergence of
Metazoa in two distinct branches leading, respectively,
to diploblasts and triploblasts. However, this topology is
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Borchiellini et al.
FIG. 1.—Deduced amino acid (aa) sequence alignment of cytoplasmic Hsp70. Partial sequences of the cytoplasmic Hsp70 from eight
diploblasts were aligned with existing Hsp70 sequences from a range of higher plants and fungi and several sequences of triploblastic animals
extracted from GenBank. A 50% consensus amino acid sequence was generated in the top line. A point indicates that the aa is identical to the
consensus; differences from the consensus are indicated. The abbreviations used in the species names are as follows: Cho. ren., Chondrosia
reniformis; Abe. hyp., Asbestopluma hypogea; Geo. cyn., Geodia cydonium; Pet. fis., Petrosia ficiformis; Rha. daw., Rhabdocalyptus dawsoni;
Eup. eur., Euplotes eurystomus; Oxy. nov., Oxytricha nova; Ara. tha., Arabidopsis thaliana; Lyc. esc., Lycopersicon esculentum; Pic. ang., Pichia
angusta; Sac. ser., Saccharomyces cerevisiae; Cae. ele., Caenorhabditis elegans; Bru. mal., Bruggia malavi; Mes. cor., Mesocestoides corti; Ech.
gra., Echinococcus granulosus; Dro. mel., Drosophila melanogaster; Cer. cap., Ceratitis capitata; Fun. qua., Funiculina quadrangularis; Eun.
cav., Eunicella cavolini; Ber. ova., Beroe ovata; Pet. mas., Petrobiona massiliana.
Monophyly of Metazoa Inferred by Hsp70 Sequences
FIG. 1
also poorly supported by bootstrap analysis. Results of
500 maximum-parsimony bootstrap replicates are shown
below the branches in figure 4.
Figure 4 shows that the Hsp70 sequences from
plants, fungi, and animals form separate monophyletic
groups taking the Alveolata as outgroup. Each monophyletic unit is supported by high bootstrap values. The
parsimony analysis reveals the same monophyletic units
651
(Continued)
as those shown in figure 4 with statistically significant
support. Results of the parsimony bootstrap analysis are
shown as a percentage indicated below each branch in
figure 4, and reveal that the monophyly of plants, fungi,
and animals is extremely robust.
The results obtained (fig. 4) from Hsp70 sequence
comparisons also indicate that animal and fungal lineages share a more recent common ancestor than either
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Borchiellini et al.
FIG. 2.—Phylogenetic analysis of Hsp70 nucleotide sequence data to determine relationships between diploblastic animals. The tree is based
upon a comparison of the nucleotide sequences at the first and second positions of codons corresponding to the amino acid alignment used in
figure 1. The topology shown here is a consensus bootstrap neighbor-joining tree obtained after 500 bootstrap replicates. The numbers above
the branches indicate the percentages of times the species are grouped together in the bootstrap trees. Distances were calculated using the
observed-divergence distance method. The root of the tree is placed within the Alveolata lineage.
does with the plant lineage with a high degree of reliability (i.e., in 91% of bootstraps). A closer affiliation
of fungal and animal species is also observed in the
maximum-parsimony tree, although the relationship is
not significantly supported by this method (80%).
Discussion
In view of the profound similarities in the organization of the epithelial and mesenchymal tissues and
connective tissue, including collagen (Garonne and Exposito 1992; Boute et al. 1996) and other molecules (for
review, see Müller 1997), between sponges and Cnidaria
plus other animals, the arguments for the monophyly of
Metazoa are very strong. However, analyses of the origin of metazoans using methods of molecular phylogeny
with rRNA as a phylogenetic index have recently revived the controversy as to whether all metazoans can
be viewed as successive offshoots within a single monophyletic unit, or different processes of cell aggregation
led to parallel radiation and different patterns of body
plan organization. The first publication of phylogenetic
trees based on partial sequences of animal 18S rRNA
(Field et al. 1988) and 28S rRNA (Christen et al. 1991)
FIG. 3.—Nucleic acid sequence phylogeny inferred from Hsp70 data. Phylogenetic analysis was performed with nucleotides at both the
first and second codon positions. The phylogenetic tree was constructed using maximum-parsimony analysis with the Alveolata lineage as the
outgroup. Numbers represent the percentages of 500 bootstrap replicates in which a given grouping was found.
Monophyly of Metazoa Inferred by Hsp70 Sequences
653
FIG. 4.—Molecular phylogenetic tree based on a comparison of nucleic acid sequences of cytoplasmic Hsp70. In order to study the
phylogenetic relationship between triploblasts and diploblasts and, more broadly, the branching orders between animals, plants, and fungi, our
sequences of ‘‘lower’’ metazoan taxa were aligned with existing Hsp70 sequences from a range of higher plants and fungi and several sequences
of triploblastic animals. The tree depicts relationships among Hsp70 sequences from 21 different species using neighbor-joining analysis with
the Alveolata lineage as the outgroup. This tree was based on nucleotide sequence at the first two codon positions. Results of 500 neighborjoining bootstrap replicates are shown above the branches. The maximum-parsimony tree for the same data set shows a similar topology. The
result of a bootstrap (maximum-parsimony) analysis is noted below the branches. Confidence levels below 50% are not indicated.
led to a revival of the idea that the two animal subkingdoms, Radiata and Bilateria, originated independently
from different flagellate protozoan ancestors. However,
these conclusions have been widely criticized (Ghiselin
1989; Walker, Bode, and Steele 1989; Patterson 1989,
1990; Lake 1990), and more recent studies based on
complete 18S rRNA sequences (Hendriks et al. 1990;
Adoutte and Philippe 1993; Wainright et al. 1993; Cavalier-Smith, Allsopp, and Chao 1994a, 1994b; CavalierSmith et al. 1996) support the monophyly of the animal
kingdom including sponges, although the bootstrap support for monophyly is very weak. However, new Hexactinellida sequences (West and Powers 1993) again
suggest that sponges may have evolved from Protozoa
independent of other animals.
Results obtained here with Hsp70 sequences show
that all the animals form a monophyletic unit. The
monophyly of animals is strongly supported by boostrap
values, both in parsimony and neighbor-joining analysis,
indicating that the animal clade is statistically significant. For the first time, high bootstrap values support
the monophyly of Metazoa (the values of 93% or 94%
are much higher than those found, for example, by
Wainright et al. [1993], by Cavalier-Smith et al. [1996],
or in any other published tree). It is also the first time
that so wide a range of diploblastic animals has been
included in phylogenetic trees.
Within the animal clade, the topology is unresolved. Results suggest that the Metazoa emerged in two
distinct branches leading, respectively, to diploblasts
and triploblasts, but this early animal dichotomy is poorly supported by bootstrap analysis. Despite the weak
bootstrap support, the topology of trees obtained in the
present work suggests that ancestors of triploblastic animals emerged much earlier than is classically assumed.
Within the diploblastic subtree, the exact branching
order between the Cnidaria, the Ctenophora, and the
three classes of Porifera remains uncertain, as shown by
the differences observed between the various methods
of tree reconstruction and the result of the bootstrap
analysis. It has been proposed that the Porifera could be
subdivided into two subphyla: the Symplasma with class
Hexactinellida, and the Cellularia with classes Calcarea
and Demospongia (Mackie and Singla 1983). However,
it has been shown in some trees (Lafay et al. 1992;
Cavalier-Smith 1993; Cavalier-Smith, Allsopp, and
Chao 1994a, 1994b; Smothers et al. 1994; CavalierSmith et al. 1996) that the Calcarea could be more close-
654
Borchiellini et al.
ly related to the Ctenophora than to the Demospongiae.
However, there is general agreement that the Porifera
can be grouped in a single phylum, although Bergquist
(1985) has proposed that the Hexactinellida could form
an independent phylum. The results presented here, despite bootstrap values that are not high, suggest a closer
relationship between Calcarea and Demospongiae classes.
In addition, in the phylogenetic analysis based on
Hsp70 sequences reported here, a closer relationship between the animals and fungi groups is suggested by the
neighbor-joining method. The high bootstrap score
(91%) of the branch point leading to the animals-fungi
clade should indicate that the affinities between these
groups are robust and significant. The parsimony tree
also favors an animals-fungi clade. However, the support
for this was not statistically robust (80%). The phylogenetic relationships between plants, animals, and fungi
have been a subject of continued debate (Cavalier-Smith
1987; Gunderson et al. 1987; Vossbrinck et al. 1987;
Gouy and Li 1989; Loomis and Smith 1990; Douglas et
al. 1991; Hendricks et al. 1991; Baldauf and Palmer
1993; Hasegawa et al. 1993; Wainright et al. 1993; Nikoh et al. 1994; Sidow and Thomas 1994; Gupta 1995;
Adoutte et al. 1996). The different trees obtained have
given contradictory topologies and have not made it
possible to determine whether the animals are more
closely related to plants or to fungi. The evolutionary
relationship between Hsp70 genes from different species
presented in this study gives additional support to the
animals-fungi clade. The most important conclusions
from Hsp70 sequence analysis are monophyly of the
Metazoa, which is supported by high bootstrap values
in both parsimony and neighbor-joining analyses, and
the specific phylogenetic relationship between animals
and fungi. This new phylogeny that recognizes the
monophyletic origin of animals and their relation to fungi provides a framework and a rational basis through
which the origins and diversification of metazoan lineages may be explored.
Acknowledgments
This work was supported by Réseau National de
Biosystématique (ACC-SV7) and the European program
MAS3-CT97-0118. We thank Claude Carré for collecting Ctenophora species, Sally Leys for collecting Rhabdocalyptus dawsoni species, and Nicolas Galtier for
kindly providing his PHYLO-WIN package.
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MASAMI HASEGAWA, reviewing editor
Accepted February 18, 1998