AMER. ZOOL., 38:878-887 (1998)
Evolution of the Multicellular Animals1
ANNA MARIE A. AGUINALDO 2 AND JAMES A. LAKE
Molecular Biology Institute and MCD Biology University of California, Los Angeles
Los Angeles, California 90095
SYNOPSIS. Molecular sequence analysis is providing new insights into the study of
metazoan relationships. The use of ribosomal RNA sequences is revising many of
the metazoan phylogenies that have been established traditionally with anatomical
and embryological data. Four new findings that seem to be well supported by
molecular data, both from the authors' laboratories and from others, are described
and discussed. First, the arthropods are members of a deep primary clade within
the protostomes and are not the sister taxa of either the annelids or the mollusks.
Second, the lophophorate animals are clearly protostomes and are contained within
a lophotrochozoan superclade including the mollusks, annelids, and many other
phyla. Third, the arthropods together with all other molting animals comprise a
second monophyletic superclade within the protostomes, the ecdysozoa. Fourth,
the platyhelminthes are contained within the lophotrochozoan superclade.
the 18S small subunit ribosomal RNA (18S
Despite more than a century of study, the rRNA). This gene is present in all organevolutionary relationships among the 30+ isms, it contains a statistically significant
phyla which comprise the multicellular an- number of bases (about 1800), and it
imals (or Metazoa) is still unresolved. evolves at a rate appropriate for studying
Much previous phylogenetic work has uti- distant relationships (Raff et al., 1994). Molized anatomical, embryological, and pale- lecular analysis of the constantly expanding
ontological data. Some of the limitations of ribosomal RNA database, while confirming
relying solely on these characters for deter- some traditional metazoan relationships, is
mining relationships are the different inter- offering a new view of the relationships
pretations of morphological features. Vary- among metazoan phyla. This paper reviews
ing identifications of homologous charac- some of these unique phylum-level relationters lead to differing phylogenetic trees (for ships that have been put forth by molecular
a review of many differing viewpoints, see analysis. Four questions addressed by moWillmer 1990). Also, many morphological lecular sequence data are: 1) What are the
interpretations are based on relatively few relationships of the coelomate phyla (aninformative traits which can relate phyla. nelids, mollusks, arthropods, echinoderms);
The use of molecular sequence data offers 2) Are lophophorates deuterostomes or proa wealth of new informative characters to tostomes; 3) What are the nearest relatives
help resolve some questions at the phylum to the arthropods; and 4) Are nematodes
level that have arisen from morphological and flatworms basal to the deuterostomes
data, but also has its own set of difficulties. and protostomes? Molecular analysis is not
Both types of data are needed and analysis without its own limitations, and these probfrom both sources are leading to new par- lems are addressed as well.
adigms.
MORPHOLOGICALLY BASED RELATIONSHIPS
The predominant molecule of choice for
our phylogenetic reconstructions has been
The multicellular animals consist of the
diploblastic (two germ layers) cnidarians
1
From the symposium Evolutionary Relationships and the triploblastic (three germ layers) biof Metazoan Phyla: Advances, Problems, and Ap- lateral animals (bilaterians). In trees derived
proaches presented at the Annual Meeting of the Society for Integrative and Comparative Biology 3-7 from morphological and from molecular sequence data, the diploblasts and triploblasts
January 1998, at Boston, Massachusetts
:
are monophyletic sister taxa (a monophyE-mail: Lakefe-mbi.ucla.edu
INTRODUCTION
878
MULTICELLULAR ANIMAL EVOLUTION
879
Traditional Tree
Articulata
^
I — Annelid
Arthropod
Eucoelomates
Protostomes
Mollusks
Lophophorates
Chordates
Bilateral
Animals
Deuterostomes
Echinoderms
Pseudocoelomates
Platyhelminthes
Cnidaria
FIG. 1. The traditional phylogenetic tree for the Metazoa (adapted from Barnes 1987) based on morphological
and embryological evidence.
letic group consists of a group which contains the last common ancestor and all its
descendants). Traditional and molecular
trees differ with respect to the placement of
phyla within the bilateral animals. An example of a traditional phylogeny frequently
found in classical invertebrate textbooks is
shown in Figure 1. In this view, the deepest
branching bilaterians were acoelomate (no
body cavity) platyhelminthes. The pseudocoelomates (false body cavity) were the
next to diverge and they preceded the radiation of the true coelomate (eucoelomate)
bilaterians which were divided into the deuterostomes and the protostomes. In this
classification, lophophorates were of uncertain affinity and were tentatively placed
somewhere between the two clades. Within
the coelomate protostomes, the mollusks
branched before the arthropods and annelids. [In protostome animals (proto = first,
stome = mouth), the mouth develops from
the first embryonic opening, the blastopore.
The best known protostome is Drosophila.
In deuterostomes (deutero = second), the
anus develops from the blastopore and the
mouth from elsewhere. The best known
deuterostome is Homo sapiens.]
This traditional phylogeny is not universally accepted within morphological community. Conflicts arise when discussing the
interrelationships between superphyletic
groupings and the placement of specific
phyla within these groups. The results of
recent morphological cladistic analyses (defining monophyletic based on shared, derived features) demonstrate the many differing topologies {e.g., Brusca and Brusca,
1990; Meglitsch and Schram, 1991; Eernisse et al., 1992; Nielsen et al, 1996). For
example, Meglistch and Schram (1991)
found that platyhelminthes diverge before
the coelomate protostomes and deuterostomes similar to the traditional phylogeny.
In contrast, Nielsen (1996) associated the
acoelomate platyhelminthes with mollusks,
annelids, arthropods and protostome phyla
using the shared, derived feature of spiral
cleavage during embryogenesis. Deuterostomes, which undergo radial cleavage,
were excluded from this group. Phylogenetic reconstructions from molecular se-
880
A. M. A. AGUINALDO AND J. A. LAKE
quence data can be utilized to distinguish
between varying topologies and can be
used to complement morphological analysis.
MOLECULAR-BASED RELATIONSHIPS
Molecular analysis is not without its pitfalls. Three different problems can confound the reconstruction of optimal phylogenetic trees. These are sequence alignment
artifacts, site-to-site variation due to varying rates of evolution within genes, and unequal rate effects caused by differing rates
of evolution among different organisms
(Lake, 1991; Raff et al, 1994). All methods
of phylogenetic reconstruction including
distance matrix, parsimony, and maximum
likelihood, can produce incorrect trees if
these artifacts are not taken into account.
Artifacts caused by greatly differing rates
of evolution in different branches of a tree
can be exhibited as the long branches
(meaning fast evolving lineages) grouping
together on a phylogenetic tree even though
the organisms are genealogically unrelated.
A recently developed reconstruction method, paralinear (LogDet) distances (Lake,
1994; Lockhart et al, 1994), is theoretically
unaffected by unequal rates in the absence
of site-to-site variation and alignment artifacts.
The studies of Field et al. (1988) pioneered the use of 18S ribosomal RNA data
to study metazoan relationships. Their work
offered an impressive database of sequences from 20 classes in 10 different animal
phyla. However, analysis of their data set
demonstrated the problems associated with
molecular reconstruction, namely due to
unequal rates of evolution in the 18S rRNA
sequences between different species (Lake,
1989, 1990) or in different regions of the
gene (Patterson, 1989). These studies and
other subsequent molecular analyses (e.g.,
Turbeville et al., 1992; Wainright et al.,
1993; Adoutte and Philippe, 1993) have
served to emphasize the need for more data
from additional organisms, the utility of using multiple methods of phylogenetic reconstruction, and the necessity of using the
most slowly evolving sequences.
• Lophophorates
O
- Echinoderms
Q.
• Chordates
I
- Arthopods
- Annelids
- Echinoderms
(0
5"
- Chordates
- Arthopods
- Annelids
- Lophophorates
FIG. 2. The topology of the Metazoa obtained via
evolutionary parsimony analysis of partial 18S rRNA
sequences (Lake, 1990) and maximum likelihood,
maximum parsimony, paralinear distance, and Kimura
two-parameter distance analyses of complete 18S
rDNA sequences (Halanych et al, 1995). The "old
view" represents the traditional morphologically-based
phylogeny and the "new view" represents the molecular-based phylogeny.
Relationships of arthropods, annelids, and
mollusks
The prevalent Articulata theory traditionally links the arthropods and annelids together based on the teloblastic formation of
their segmented body plans. Molecular analyses, however, have not supported this
view. Early analysis of 18S rRNA data
showed that arthropods arose separately
from a coelomate protostome grouping that
included annelids, mollusks, sipunculans,
brachiopods, and pogonophorans, although
the arthropods were themselves found to be
paraphyletic (Lake, 1990). Deuterostomes
were found to be monophyletic, supporting
the traditional viewpoint, but arthropods did
not fit the traditional view and diverged before annelids and mollusks (Lake, 1990;
Fig. 2). Molecular analyses with additional
and complete 18S rDNA sequences have
since supported these results (Turbeville et
al, 1992; Adoutte and Philippe, 1993; Halanych et al, 1995). A combined analysis of
MULTICELLULAR ANIMAL EVOLUTION
morphological and molecular data also supports an annelid-mollusk lineage (Kim et
al, 1996) as does a cladistic treatise (Eernisse et al, 1992).
Lophophorates—Protostomes or
deuterostom.es
Lophophorates, comprised of the brachiopods, bryozoans, and phoronids, are
linked together by the presence of a circular
or horseshoe-shaped, tentacular ringed
mouth (the lophophore). They have been allied with the deuterostomes because of the
lophophore-like structure (also found in
pterobranch hemichordates), a tricoelomic
body arrangement, modified radial cleavage, coelom arising by enterocoely (brachiopods), the formation of the anus from
the blastopore (in some brachiopods), and
a U-shaped adult digestive cavity (Ruppert
and Barnes, 1994; Halanych et al, 1995).
However, they also exhibit protostome affinities because of the presence of chitin
(but see Willmer, 1990, regarding the possible presence of chitin in cephalochordates), lack of sialic acids, the presence of
larval protonephridia (in phoronids), and
the formation of the mouth from the blastopore (in bryozoans and phoronids) (Ruppert and Barnes, 1994; Willmer, 1990).
A molecular analysis using complete 18S
ribosomal DNA sequences from the three
representative lophophorate phyla (bryozoans, phoronids, and brachiopods) placed
them among the protostomes (Halanych et
al, 1995; see also Macky et al, 1996). The
topology of the relationship is shown in
Figure 2. Using four different reconstruction algorithms, the distance matrices of
paralinear distances and Kimura two-parameter distances, parsimony and maximum
likelihood, it was found that bryozoans,
brachiopods, and phoronids formed a clade
with mollusks and annelids to the exclusion
of arthropods. This clade, called the Lophotrochozoa, includes the ancestor of lophophorate taxa and taxa which arise via a
trochophore larva, and all other descendants. These results confirm preliminary
molecular analyses using a single incomplete brachiopod 18S rRNA sequence that
placed the inarticulate brachiopod within a
mollusk-annelid lineage within the proto-
881
stomes (Field et al, 1988; Ghiselin, 1988;
Lake, 1989, 1990; Patterson, 1989. Although the similar lophophore feeding apparatus of the lophophorates had suggested
a monophyletic origin for these organisms,
the 18S rDNA analyses found them to be
paraphyletic with the bryozoans diverging
before the rest of the Lophotrochozoans
(Halanych et al, 1995). Other studies using
additional sequences have also exhibited
protostome affinities and the polyphyly of
the lophophorates (Mackey et al, 1996).
Arthropods and other molting animals
Once it became clear that the arthropods
are separated from many of the protostome
phyla, it became important to determine if
any other protostome groups were close relatives of the arthropods. Some proposed
sister taxa of the arthropods included the
tardigrades and the onychophorans. Like arthropods, they both have a greatly reduced
coelom, a hemocoel serves as the circulatory system with a dorsal, ostiate heart, they
periodically molt, and they lack locomotory
cilia (Brusca and Brusca, 1990). We sought
to determine the relationships of these phyla to arthropods using 18S rDNA sequences
and uncovered some surprising alliances
(Aguinaldo et al, 1997). Using three different distance methods and parsimony, not
only did onychophorans and tardigrades
group with the arthropods, but it was found
that all molting phyla formed a monophyletic group. These phyla include the nematodes, kinorhynchs, nematomorphs, and
priapulids (see Fig. 3). [Loriciferans, which
also molt, are presumed to also be close arthropod relatives. Since they are so rare,
only a single living loriciferan larva has
been observed (Kristensen, 1991), they
could not be sequenced.]
The close relationship of the nematodes
to the arthropods was an unexpected result.
Nematodes are traditionally associated with
the pseudocoelomates, a group of taxa including gastrotrichs, rotifers, nematomorphs, kinorhynchs, priapulids, acanthocephalans that have been thought to share
a body cavity without a peritoneal lining
(Raff et al, 1994). This superphyletic group
was usually placed between the acoelomates and the coelomates. However, cladis-
882
A. M. A. AGUINALDO AND J. A. LAKE
Priapulids
Kinorhynchs
Ecdysozoa
Nematomorphs
Onychophorans
Nematodes
Tardigrades
Arthropods
Protostomes
(Crustaceans)
Arthropods
(Insects)
Arthropods
(Chelicerates)
Arthropods
(Myriapods)
Bilateral
Animals
Lophotrochozoa
V
Rotifer
Annelids
(Oligochaetes)
Brachiopods
Annelids
(Polychaetes)
Mollusks
Echinoderms
Cnidarians
FIG. 3. The consensus tree from paraiinear distance, maximum parsimony, Kimura two-parameter distance, and
Jukes-Cantor distance analyses of 18S rDNA sequences showing the relationships of the molting metazoans
(adapted from Aguinaldo et ah, 1997). Major clades are denoted by filled circles.
tic analyses of morphological features (Lorenzen, 1985; Eernisse et al., 1992) and molecular analysis (Winnepenninckx et al,
1995) have raised questions about the
monophyletic grouping of these taxa. Previous molecular analyses placed the nematodes deep within the tree, before the protostome/deuterostome bifurcation (Philippe
et al, 1994; Winnepenninckx et al, 1995).
However, these authors recognized that the
rapid evolution of nematode sequences
could have made these results suspect. As
discussed above, unequal rate effects
caused by rapidly evolving sequences can
artifactually place long branches together
(in this case the nematodes with the longbranched outgroups). Our own analysis, using two rapidly and one reasonably slowly
evolving nematode sequences, found that
nematodes branch from the base of the bilateral animals when all three sequences are
analyzed (Fig. 4A), but branch high within
the protostomes as the sister taxon of the
arthropods when only the slowly evolving
nematode sequence is included (Fig. 4B)
(Aguinaldo et al., 1997).
To limit the effects due to unequal rates
of evolution, we surveyed nearly 50 complete 18S rDNA sequences (including unpublished sequences by Jim Garey and his
coworkers and those available in the databases) and chose the slowest evolving sequences for subsequent phylogenetic analysis (see Table 1 for an example of some
of the rates used to select sequences for
subsequent study). Analyses of these slowly
evolving sequences by several methods produced trees containing a clade of all molting animals (Aguinaldo et al, 1997). We
named this group the Ecdysozoa. Denning
synapomorphies for this clade include molting by ecdysis and lack of motile cilia. A
883
MULTICELLULAR ANIMAL EVOLUTION
Priapulid
Arthropod
(Chelteerate)
Arthropod
(CnMtac*an)
Arthropod
(Inswt)
Mollusk
(Blvilve)
Mollusk
(Polypi acophoran)
Polychaete
Echlnoderm
Nematode
(Trtchlrtollaj
Nematode
(SmuiyytoMuj
Nematode
(CmnoriiibdOs)
Cnidarian
B
Priapulid
Arthropod
(Chalicerite)
Nematode
(Trichlnatb)
Arthropod
(Cnntacean)
Arthropod
(lns«d)
Mollusk
(Bivalve)
Mollusk
(Polyplacophoran)
Polychaete
Echlnoderm
Cnidarian
FIG. 4. Phylogenetic analysis of 18S rDNA illustrating the effects of unequal rate biases on nematode placement
(adapted from Aguinaldo et al., 1997). In A, both rapidly (Caenorhabditis and Strongyloides) and slowly evolving (Trichinella) nematode sequences are included in the analysis; the nematodes branch early within the tree,
before the deuterostome-protostome divergence. In B, using only the slowly evolving Trichinella sequence, the
nematode now branches within the protostome clade as the sister taxon to the arthropods.
previous cladistic analysis supported a
clade of some, but not all, molting animals
since priapulids were not contained within
the group and nematomorphs were not studied (Eernisse et al., 1992). These results
suggest that the protostomes are comprised
of two distinct clades, the arthropod-related
group exclusively made up of molting animals and a lophotrochozoan group containing non-molting protostome animals.
The placement of rotifers within the Lophotrochozoa and the nematodes, nematomorphs, priapulids, and kinorhynchs within
the Ecdysozoa contradicts the monophyly
of pseudocoelomates. This is consistent
with the paraphyletic origins based on 18S
rDNA previously found by Winnepenninckx et al. (1995).
Flatworms—Basal to protostomes and
deuterostomes ?
Since nematodes differed in their phylogenetic placements depending on the rates
of the sequences chosen, we applied the
concept of using the slowest evolving sequences to study the affinities of the platyhelminthes. According to the traditional
view, an acoelomate, flatworm-like ancestor
is thought to have given rise to the bilaterian animals (Hyman, 1951; Barnes, 1987),
and the platyhelminthes, with their acoelo-
884
A. M. A. AGUINALDO AND J. A. LAKE
TABLE 1. Nucleotide substitution rates of metazoan J8S rDNA.*
Arthropods and relatives
Nematoda
Onychophora
Tardigrada
Nematomorpha
Arthropoda
Priapula
Chordata
Echinodermata
Ctenophora
Cnidaria
Strongyloides
Caenorhabditis
Nippostrongylus
Trichinella
Euperipatoides
Milnesium
Macrobiotus
Gordius
Anemia
Panulirus (crustacean)
Drosophila
Crossodonthina
Tenebrio (insect)
Scolopendra (myriapod)
Androctonus
Eurypelma (chelicerate)
Priapulus
Outgroups
Lampetra
Branchiostoma
Slrongylocentrolus
Antedon
Mnemiopsis
Anemonia
Tripedalia
0.192 Jt
0.187 1t
0.137 it
0.110 it
0.090 dt
0.079 it
0.079 1t
0.068 it
0.068 it
0.065 ib
0.121 1t
0.056 dt
0.048 it
0.043 dt
0.046 :t
0.038 ±
0.040 ±
0.014
0.013
0.010
0.010
0.009
0.008
0.009
0.007
0.007
0.008
0.011
0.007
0.006
0.006
0.006
0.005
0.005
0.065 dt 0.007
0.059 dt 0.006
0.043 dt 0.006
0.040 it
0.130 di
0.101 ±
0.100 ±
0.005
0.011
0.009
0.009
* Adapted from Aguinaldo el al., 1997.
Bold-faced taxa represent some of the slowest evolving representative taxa selected for phylogenetic analysis.
mate condition, are considered one of the
first to diverge from this ancestor, forming
the sister taxa to the Bilateria. Another view
places the platyhelminthes as the sister taxa
to the spiral cleaving protostomes based on
similar cleavage patterns. This view is supported by Brusca and Brusca (1990) and
Nielsen (1995). Other possibilities such as
the "archicoelomate" theory of Siewing
(1980) are reviewed elsewhere (Willmer,
1990).
Using a slowly evolving flatworm sequence, our molecular analyses (Aguinaldo
et al., 1997) showed that platyhelminthes
are contained within the lophotrochozoan
clade (see Fig. 5). The cladistic analyses of
Eernisse et al. (1992) also associated platyhelminthes with protostomes that developed via a trochophore larva. In contrast to
our results, previous molecular analyses of
flatworm sequences have placed them as
sister taxa to the bilaterians (Philippe et al.,
1994; Winnepinnenckx et al, 1995). However, the sequences used in those studies exhibited long branches (the result of fast
evolving lineages) and the deep placement
could be attributed to unequal rate effects.
A recent molecular analysis produced a
new, extensive 18S rDNA flatworm data
set, but yielded incongruent results for the
placement of platyhelminthes (Carranza et
al., 1997). In that study, distance methods
found the flatworms as sister group to the
bilaterians, maximum parsimony grouped
the flatworms with deuterostomes, and
maximum likelihood grouped flatworms
with all other protostomes. However, the inconclusive findings may have resulted from
the biased sampling number of flatworm sequences relative to the other metazoans (16
flatworm sequences compared with 13-16
other metazoans) and the extremely long
branches exhibited by the majority of the
flatworm lineages. In contrast, another recent 18S rDNA molecular analysis found
the platyhelminthes among a lophotrochozoan assemblage (Balavoine, 1997) with
both distance and maximum parsimony
methods, consistent with our findings.
These results suggest that the acoelomate
MULTICELLULAR ANIMAL EVOLUTION
885
Platy helminthes
Lophotrochozoa
Rotifer
Annelids
(Oligochaetes)
Annelids
(Polychaetes)
Brachiopods
Protostomes
Mollusks
(Polyplacophorans)
Mollusks
(Bivalves)
Nematodes
Bilateral
Animals
Nematomorphs
Arthropods
(Chellcerates)
Arthropods
(Myriapods)
Arthropods
(Insects)
Arthropods
(Crustaceans)
Echinoderms
Cnidarians
FIG. 5. The consensus tree derived from paralinear distance, maximum parsimony, Kimura two-parameter
distance, and Jukes-Cantor distance analyses of 18S rDNA sequences showing the relationships of platyhelminthes within the Metazoa (adapted from Aguinaldo et al., 1997). Major clades are denoted by filled circles
and arrows.
triploblasts are derived from coelomate protostomes through a subsequent loss of the
coelom.
DISCUSSION
The molecular phytogenies determined in
our studies differ from the traditional view
in several ways. Figure 6 illustrates some
of these changes. The most significant feature is the separation of protostomes into
two groups, an arthropod-related clade exclusively composed of animals that molt,
and a lophotrochozoan clade exclusively
containing non-molting animals (see Fig. 3
and 5 for specific phyla affinities). Both of
these clades contain representative pseudocoelomate phyla and the acoelomate platyhelminthes fall within the lophotrochozoa.
All members of the arthropod-related
clade undergo ecdysis, hence their name,
the Ecdysozoa. In addition, all members
lack locomotory cilia. (A few lophotrochozoans, chaetognaths and acanthocephalans,
also lack them.) Given the observed tree topology and these common structural features, this raises the possibility that ecdysis
and the many cellular modifications associated with it may have been derived only
once within this clade. Several members of
this clade (nematodes, nematomorphs, kinorhynchs, priapulids, and loriciferans) also
share the feature of a spiny, anterior introvert (Nielsen, 1995). Additionally, the success of the ecdysozoa, containing several
speciose phyla (for example the arthropods
and the nematodes), is notable. Furthermore
some members of this superphylum, for example arthropods and priapulids, are numerically prominent members of the Burgess shale faunas (Conway Morris, 1993),
886
A. M. A. AGUINALDO AND J. A. LAKE
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Metazoan phylogeny illustrating major changes from the old view (A) to the new view (B).
indicating their success even in the early
stages of the evolution of bilateral life.
The lophotrochozoan clade includes nonmolting phyla which have a lophophore
feeding apparatus (bryozoans, phoronids,
and brachiopods) or develop via a trochophore larvae (mollusks and annelids but
also sipunculans, echiurans, and pogonophorans). Additionally, several pseudocoelomate phyla (rotifers, gastrotrichs, acanthocephalans [Winnepenninckx et al., 1995;
Aguinaldo et al., 1997]), the platyhelminthes, and the nemerteans (Aguinaldo et al,
1997; Balavoine, 1997) are included. Hox
gene data gives added support for the inclusion of platyhelminthes as a member of
the Lophotrochozoa (Balavoine, 1997).
Representative lophotrochozoans, mollusks
and brachiopods, are also well represented
in the Cambrian fossil record (Conway
Morris, 1993)
Studies from our labs and others illustrate the utility of selecting slow evolving
sequences for molecular reconstruction to
detract from incorrect phylogenies caused
by unequal rate effects. Sampling of other
molecules and of additional slow evolving
taxa will be necessary to further test the
phylogeny we have presented. We hope that
the new insights gained through molecular
phylogenetics can be integrated with studies
of morphology, development, paleontology,
and life history studies and lead to an improved understanding of the evolution of
multicellular life.
REFERENCES
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Basel.
Aguinaldo, A. M. A., J. M. Turbeville, L. S. Linford,
M. C. Rivera, J. R. Garey. R. A. Raff, and J. A.
Lake. 1997. Evidence for a clade of nematodes,
arthropods, and other moulting animals. Nature
387:489-493.
Balavoine, G. 1997. The early emergence of platyhelminths is contradicted by the agreement between
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MULTICELLULAR ANIMAL EVOLUTION
Barnes, R. D. 1987. Invertebrate zoology, 5th edition.
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Brusca, R. C. and G. J. Brusca. 1990. Invertebrates.
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Corresponding Editor: Douglas H. Erwin