Are the Platyhelminthes a Monophyletic Primitive Group? An Assessment
Using 18s rDNA Sequences
Salvador
Carranza,
Jaume Bagu&i, and Marta Riutort
Departament
de Genetica,
Facultat de Biologia.
Universitat
de Barcelona,
Spain
In most zoological textbooks, Platyhelminthes
are depicted as an early-emerging
clade forming the likely sister
group of all the other Bilateria. Other phylogenetic proposals see them either as the sister group of most of the
Protostomia or as a group derived from protostome coelomate ancestors by progenesis. The main difficulty in their
correct phylogenetic placing is the lack of convincing synapomorphies
for all Platyheh-ninthes, which may indicate
that they are polyphyletic.
Moreover, their internal phylogenetic
relationships
are still uncertain. To test these
hypotheses, new complete 18s rDNA sequences from 13 species of “Turbellaria” have been obtained and compared
to published sequences of 2 other “Turbellaria,” 3 species of parasitic Platyhelminthes,
and several diploblastic and
deuterostome and protostome triploblastics. Maximum-parsimony,
maximum-likelihood,
and neighbor-joining methods were used to infer their phylogeny. The results show the order Catenulida to form an independent earlybranching clade and emerge as a potential sister group of the rest of the Bilateria, while the rest of Platyhelminthes
(Rhabditophora),
which includes the parasites, form a clear monophyletic group closely related to the protostomes.
The order Acoela, morphologically
considered as candidates to be ancestral, are shown to be fast-clock organisms
for the 18s rDNA gene. Hence, long-branching
of acoels and insufficient sampling of catenulids and acoels leave
their position still unresolved and call for further studies. Within the Rhabditophora,
our analyses suggest (1) a
close relationship between orders Macrostomida and Polycladida, forming a clear sister group to the rest of orders;
(2) that parasitic platyhelminthes
appeared early in the evolution of the group and form a sister group to a stillunresolved clade made by Nemertodermatida,
Lecithoepitheliata,
Prolecithophora,
Proseriata, Tiicladida, and Rhabdocoela; and (3) that Seriata is paraphyletic.
Introduction
The origin of Bilateria (organisms
which display
bilateral symmetry
and clear anteroposterior
polarity)
can be considered the most important unsolved problem
in systematic biology. These animals also share the characteristic of possessing three clearly distinct cell layers
(i.e., they have a true mesoderm); hence, they are collectively called triploblastics.
Historically, the presence/
absence of a true coelom (i.e., a system of cavities within the mesoderm), and, hence, whether the most primitive body form was acoelomate,
pseudocoelomate,
or
coelomate, has been the main issue in discussion of the
origin of the Bilateria. The most classical view, adopted
in the majority of zoological textbooks, sees the acoelomate Platyhelminthes
as an early-emerging
clade
forming the likely sister group of all the other Bilateria,
which themselves would be divided into two coelomate
supergroups,
protostomes
and deuterostomes
(Hyman
1951; Salvini-Plawen
1978; fig. 1A). Since egg cleavage
in Platyhelminthes
is spiral, as in most protostomes,
while it is radial in most deuterostomes,
this proposal
implies several modifications
of early embryogenesis
in
the deuterostome
line with respect to the protostome
one. To skip that problem, another phylogenetic
scheme
sees two Bilateria supergroups:
the “Spiralia”
on the
one hand, including the Platyhelminthes,
and the “Radialia” on the other (fig. 1B). Under that scheme, the
coelom must therefore have originated twice, once in
Key words: 18s rDNA, Platyhelminthes,
monophyletic
primitive group.
phylogeny,
metazoan,
Address for correspondence and reprints: Dr. Marta Riutort, Departament de Genktica, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 0807 1 Barcelona, Spain. E-mail: [email protected].
Mol. Biol. Evol. 14(5):485497.
1997
0 1997 by the Society for Molecular Biology
and Evolution.
ISSN: 0737-4038
the Spiralia (after the split of one branch from Platyhelminthes)
and once in the Radialia (Ax 1987; Brusca
and Brusca 1990). A third view is based on the idea that
the gastral pouches of coelenterates
are homologous
with the gastral pouches (enterocoels)
that give rise to
the coeloms in deuterostomes.
Therefore, features of
deuterostome
development
are assumed to be primitive
among Bilateria. In this scheme, protostome developmental features are derived, coelom is of early origin,
Platyhelminthes
are considered derived from a coelomate ancestor by progenesis (Rieger 1985) or by reduction of coelomic cavities in the adult (Remane, Starch,
and Welsch 1980), and the hypothetical ancestor of the
Bilateria would be an “archicoelomate”
(Siewing 1980)
(fig. 10
Knowledge of the actual phylogenetic
position of
Platyhelminthes
is paramount to decide among these alternatives. Platyhelminthes,
or flatworms, display a variety of body forms and are succesful inhabitants of a
wide range of environments.
The majority of their
20,000 extant species are parasitic (classes Trematoda,
Monogenea
and Cestoda). The free-living forms (class
Turbellaria)
are primarily epifaunal or infaunal inhabitants of the marine and freshwater benthos, but marine
and freshwater pelagic and terrestrial forms also occur.
The free-living
forms range from less than 1 mm to
about 50 cm long. Some parasites (tapeworms) may attain lengths of several meters. Major diagnostic features
of the phylum (synapomorphies)
are disputed. Ehlers
(1985, 1986; see also Ax 1987) has proposed as autapomorphies some features of protonephridia,
multiciliation in epidermal cells, and absence of mitosis in epidermal and other somatic cells, but such views have
been contested (Smith, Tyler, and Rieger 1986; Rohde
1990). This casts doubt on the monophyly
of the Pla485
_
~_. ---
486
Carranza
*
-
et al.
Cnidaria
Platyhelminthes
Protostomia
Bilateria
Deuterostomia
Cnidaria
Platyhelminthes
‘Spiralia’
Protostomia
Deuterostomia
‘Radialia’
Platyhelminthes
Protostomia
Bilateria
Deuterostomia
FIG. 1.-Conflicting
traditional phylogenies 01nl the origin of Platyhelmmthes and the acoelomate condition. A, evolutionary tree based
on the assumption that the acoelomate condition is primitive within
the triploblasts. Under this view, Platyhelminthes
form the sister group
of all the other Bilateria, which themselves would be divided into two
coelomate supergroups, protostomes and deuterostomes
(after Hyman,
1951). B, An evolutionary
tree based on a very early splitting of Bilateria into two supergroups,
the “Spiralia,”
including the Platyhelminthes, and the “Radialia.”
In this scheme, the acoelomate condition
is primitive within the triploblastic spiralians, making Platyhelminthes
the first descendant group of this lineage but not the sister group of
the Bilateria (after Ax 1987; Brusca and Brusca 1990). C, An evolutionary tree based on the proposal that the acoelomate condition arose
through neoteny from developmental stages of protostomes prior to the
embryonic appearance of coelomic cavities. In this scheme, coelomic
cavities (enterocoels)
in deuterostomes
are homologous
to gastral
pouches of the diblastic coelenterates,
protostome coelomic cavities
(schizocoels) are derived, and the hypothetical ancestor of the bilateria
would be an “archicoelomate”
(after Sewing 1980; Rieger 1985).
tyhelminthes.
Indeed, the common denominator
of all
recently proposed phylogenetic
schemes for the platyhelminths is recognition
of three clearly monophyletic
groups: one containing the Acoela and Nemertoderma-
tida (“Acoelomorpha”
of Ehlers 1985, 1986), one containing the Catenulida, and the third containing all other
turbellarian
orders together with the parasitic classes
(“Rhabditophora”
of Ehlers 1985, 1986) (fig. 2A). As
there are no convincing
synapomorphies
for all Platyheminthes,
this may indicate that Platyhelminthes
are
polyphyletic
and that Catenulida,
Acoelomorpha,
and
Rhabditophora
may be unrelated taxa. As first suggested
by Ehlers (1985, 1986) and Ax (1987), this means that
the free-living
class, the Turbellaria,
are paraphyletic.
Indeed, features defining it (free-living life style and the
body covered by a ciliated epidermis)
are plesiomorphies (Ehlers 1985, 1986; Ax 1987). Finally, affinities
between the different “turbellarian”
orders, as well as
whether a parasitic group is monophyletic
or polyphyletic and, if the former, which is its closest “turbellarian” taxon, are uncertain and a matter of debate. Although phylogenetic
schemes, based mainly on morphological characters
and electron
microscope
features,
have been proposed (Karling 1974, fig. 2b; Ehlers 1985;
Rohde 1990), presently available information cannot decide among them.
Morphological
and embryological
comparisons between Platyhelminthes
and presumed close phyla (e.g.,
gnathostomulids,
pseudocoelomates,
gastrotrichs, acanthocephalans, rotifera) have so far been unable to answer
the main question on the phylogenetic
position of Platyhelminthes
as related to the rest of the Bilateria and
its mono- or polyphyletic
status. This is because these
phyla share few informative homologous anatomical or
embryological
features and because it is very difficult
to distinguish homologous
similarity (homology) from
convergent
or parallel similarity (analogy). Moreover,
most Platyhelminthes
are small and soft-bodied organisms which lack a fossil record. Sequence data obtained
from ribosomal RNA or DNA offer an important new
source of informative characters for inferring high-level
phylogenetic relationships for many taxa and provide an
independent
test of hypotheses based on morphological
characters (Woese 1987; Field et al. 1988; Turbeville,
Field, and Raff 1992; Adoutte and Philippe 1993; Riutort et al. 1993; Smothers et al. 1994; Winnepenninckx,
Backeljau and De Wachter 1995; Friedrich and Tautz
1995; Bridge et al. 1995). The main reasons for using
18s rDNA or rRNA have been repeatedly reviewed
(Woese 1987; Sogin 1991).
18s rDNA sequences, either partial or complete,
from some species of Platyhelminthes
have been used
either in general molecular analysis of animal evolution
(Field et al. 1988; Riutort et al. 1993; Adoutte and Philippe 1993; Philippe, Chenuil, and Adoutte 1994; Wada
and Satoh 1994) or in specific studies of particular phyla
or groups of phyla (e.g., protostome worms: Winnepenninckx, Backeljau,
and De Wachter 1995; Myxozoa:
Smothers et al. 1994; Dicyemid Mesozoa: Katayama et
al. 1995; Nemertini: Turbeville, Field, and Raff 1992;
Arthropoda: Turbeville et al. 1991). In most studies, Platyhelminthes
appear as the sister group of Bilateria. In
others, they cluster with nematodes, acanthocephalans,
and some protostomes in ill-defined paraphyletic groups.
Ingroup
studies of Platyhelminthes
phylogeny
have
The Phylogenetic
Status of Platyhelminthes
487
B
Catenulida
I Catenulida
Catenulida
Acoela
Nemertodermatida
I
Acoelomorpha
Acoela
Nemertodermatida
Macrostomida
Macrostomida
Polycladida
Polycladida
Lecithoepitheliata
Prolecithophora
Proseriata
Lecithoepitheliata
Rhabditophora
Rhabdocoela
Neodermata
Tricladida
Rhabdocoela
Prolecithophora
l’gphloplanoitla
Rhabdocoela
Proseriata
Dalgellioida
Neodermata
Tricladida
FIG. 2.-A, Phylogeny of Platyhelminthes
according to Ehlers (1985). B, Evolutionary
Turbellaria and the parasitic platyhelminths
(after Karling 1974).
mainly concerned
parasitic groups (Baverstock
et al.
1991; Blair 1993; Rohde et al. 1993, 1995) and a few
turbellarian orders (Riutort et al. 1992, 1993; Katayama
et al. 1993; Rohde et al. 1993, 1995). The main conclusions are that all the major parasitic groups constitute a
monophylum,
that class Trematoda
is monophyletic
whereas Monogenea may be paraphyletic, that there are
contradictory
views as regards the closest free-living
turbellarian to parasitic groups, and that Platyhelminthes
may be polyphyletic
(with acoels forming a separate
clade; Katayama et al. 1995). Most of these data, however, should be taken very cautiously because most studies used only partial 18s sequences and because “lower” platyhelminth
orders (Catenulida,
Nemertodermatida, Lecithoepitheliata,
and Macrostomida)
either were
not included or were represented by a single species.
We report here the complete 18s rDNA sequences
of 13 species of free-living
“Turbellaria”
belonging to
10 orders. Published complete sequences of two other
free-living
turbellaria
and three parasitic species were
also included in some of the analyses. Sequence data
were analyzed with maximum-parsimony,
distance-matrix, maximum-likelihood,
and pattern of resolved nodes
(PRN) methods. Our main focus has been to test: (1)
the monophyletic
or polyphyletic
status of the Platyhelminthes,
(2) whether Platyhelminthes
can be considered the sister group of the rest of bilaterians,
(3) the
internal relationships
among the extant “turbellarian”
orders, and (4) which is the closest free-living representative of the parasitic groups.
Materials
Organisms
and Methods
Organisms were chosen to provide representatives
of the 11 orders of Turbellaria.
In addition, published
Platyhelminthes
sequences
were used. Species represented are shown in table 1, and the 13 species for which
new sequence data were collected are indicated. Because
there is no consensus
as to the sister group of Platyhelminthes (see Introduction),
we used as outgroup the
tree depicting
the relationships
among the orders of
choanoflagellate
Sphaeroeca volvox. Since diploblastics
have been clearly shown to be the sister group of all
Triblastics, the cnidarian Anemonia sulcatu, the ctenophore Beroe cucumis, and the placozoan Trichoplux adhaerens were also included in the analysis. Representatives of Deuterostomia
were the starfish Asterias amuBrunchiostoma jloridue, and
rensis, the cephalochordate
the amphibian Xenopus laevis, while Aphonopelma
sp.
(Arthropoda,
Aracnida),
Odiellus troguloides (Arthropoda, Arachnida), Eisenia fetidu (Annelida, Oligochae(Annelida,
Polychaeta),
Liolota), Lunice conchilega
phuru juponica
(Mollusca,
Polyplacophora),
Crussostrea virginica (Mollusca, Bivalvia), and Prostoma eilhardi (Nemertea)
were used as representatives
of
Protostomia. Because recent analyses indicate that pseudocoelomates,
namely the phyla Nematoda and Nematomorpha, may form the sister group of the rest of Bilateria, and since Gastrotricha and Acanthocephala
appear as a likely sister groups of Platyhelminthes
(Winnepenninckx
et al. 1995; Doolittle
et al. 1996),
sequences from the nematomorph
Gordius aquaticus,
the gastrotrich
Lepidodermella
squammata,
and the
acanthocephalan
Moliniformis moliniformis were included in some of the analyses. All nonplatyhelminth
species
are listed in table 2.
DNA Extraction,
Amplification,
and Sequencing
High-molecular-weight
DNA was purified according to a modification
(Garcia-Fernandez,
Bagufia, and
Sal6 1993) of the guanidine isothiocyanate
method initially described for RNA (Chirgwin et al. 1979). The
entire length of the 18s rDNA was amplified in two
fragments of approximately
equal length by the polymerase chain reaction (Saiki et al. 1985) using the primers: 1E TACCTGGTTG
ATCCTGCCAG
TAG; 5R,
CTTGGCAAAT
GCTTTCGC;
5F, GCGAAAGCAT
TTGCCAAGAA;
and 9R, GATCCTTCCG
CAGGTTCACC TAC. The resulting fragments were either blunt
end cloned and sequenced as described elsewhere (Carranza et al. 1996) or directly sequenced (Nemertinoides,
488
Carranza
et al.
Table 1
List of Platyhelminth Species Used in this Study and
GenBank Accession Numbers
Accession
Number
Table 2
List of Nonplatyhelminth Species Included in this Study
and GenBank Accession Numbers
Name in
Figures
Class Turbellaria
Accession
Number
Phylum
Order Tricladida
Triclad- 1
Triclad-2
Xenopus laevis
Monocelis lineata* . . . . . . . . . . U45961
Archiloa rivularis” . . . . . . . . . . U70077
Proseriate- 1
Proseriate-2
Order Lecithoepitheliata
Geocentrophora
Mesocastrada
sp.*
Lecithoepitheliate
. . . . . . . . . . U70082
Rhabdocoel
Order Nemertodermatida
Nemertinoides
. . . . U70084
Nemertodermate
. . . . . . . . . . . . . U70086
Prolecithophoran
elongatus*
Order Macrostomida
tuba*. . . . . . . . . U7008 1
lineare* . . . . . . . U70083
Macrostomum
Microstomum
Macrostomid- 1
Macrostomid-2
Order Polycladida
tigrina* . . . . . . . . . . U70079
multitentaculata
. . . D17562
Polyclad- 1
Polyclad-2
. . . . . . . U70085
Catenulid
Convoluta pulchra” . . . . . . . . . U70078
Convoluta naikaiensis . . . . . . . D17558
Acoel- 1
Acoel-2
Stenostomum
leucops”
Arthropod-
Phylum Annelida
Class Clitellata
Eisenia fetida . . . . . . . . . . . . . . . X79872
Class Polychaeta
Lanice conchilega . . . . . . . . . . . X79873
Phylum
Cestode
Subclass
Phylum
Aspidogastrea
Lobatostoma
manteri
. . . . . . . . L 169 11
Phylum
Trematode-
1
Digenea
Phylum
Schistosoma
NOTE.--New
mansoni.
. . . . . . . M62652
Mesocastrudu,
and Archiloa).
pletely sequenced.
Sequence
Alignment
Both chains
and Phylogenetic
Nemertine
. . . U29198
Gastrotrich
. . . . . . . . . . . X87985
Nematomorph
Acanthocephala
moliniformis. . . . . 219562
Acanthocephalan
Placozoa
. . . . . . . . L 10828
Placozoan
. . . . . . . . . . . X53498
Cnidarian
adhaerens
Cnidaria
sulcata.
Ctenophora
Trematode-2
Phylum
Annelid-
. . . . . . . . . . . U29494
squammata.
Beroe cucumis
sequences reported in this paper are marked with an asterisk.
Annelid- 1
Nematomorpha
Anemonia
Order Schistosomida
Arthropod-2
Gastrotricha
Trichoplax
Order Aspidobothria
Subclass
eilhardi
Moliniformis
Class Trematoda
1
Nemertini
Lepidodennella
Phylum
. . . . U27015
Mollusc- 1
Arthropoda
Gordius aquaticus
granulosus.
. . . . . . . . . X702 10
Class Arachnida
Order Opiliones
Odiellus troguloides. . . . . . . . . . X8 144 1
Order Araneae
Aphonopelma sp. . . . . . . . . . . . . X 13457
Class Cestoda
Echinococcus
Echinoderm
Mollusc-2
Phylum
Order Cyclophyllida
Vertebrate
Mollusca
Prostoma
Order Acoela
. . . . . . . . . . . . . . X04025
. . . . . . . . X603 15
Phylum
Order Catenulida
. . . . . . . M9757 1 Cephalochordate
Echinodermata
Asterias amurensis
. . . . . . . . . . . D 14358
Class Polyplacophora
Liolophura japonica.
Class Bivalvia
Crassostrea virginica.
Phylum
Order Prolecithophora
sp.*.
Phylum
Phylum
. . . . . . . . U70080
sp.*
Order Rhabdocoela
Discocelis
Planocera
Cephalochordata
Branchiostoma j7oridae
Subphylum Vertebrata
Order Proseriata
Urastoma
Chordata
Subphylum
Crenobia alpina* . . . . . . . . . . . M58345
Dendrocoelum lacteum* . . . . . M58346
Name in
Figures
. . . . . . . . . . . . . . D 15068
Ctenophore
Sarcomastigophora
Sphaeroeca
volvox.
. . . . . . . . . . 234900
Choanoflagellate
were com-
Analyses
Sequence data were aligned by hand with the help
of a computer editor. Alignment
gaps were inserted to
account for putative length differences between the sequences. A secondary-structure
model (Gutell et al.
1985) was used in order to optimize alignment of homologous nucleotide
positions, resulting in a total of
1,322 positions that could be used in the phylogenetic
analyses (675 being variable and 428 being parsimonyinformative when the 33 species are compared).
Distance analyses were calculated using the PHYLIP program package v. 3.52 (Felsenstein
1993) and
MUST v. 1.0 (Philippe 1993). A distance matrix of the
aligned sequences
was generated using the program
DNADIST and corrected with the two-parameter
method of Kimura (1980). The distances were then converted
to phylogenetic
trees using FITCH (Fitch and Margoliash 1967) and the neighbor-joining
(NJ) method of Saitou and Nei (1987) provided by the NEIGHBOR
program. Bootstrap resampling (Felsenstein
1985) was accomplished
with the use of the programs SEQBOOT
(1,000 replicas)
and CONSENSE.
FASTDNAML
v.
1.1. la (with global rearrangements
and reordering of
species) was used for maximum-likelihood
analyses
(Felsenstein
1981; Olsen et al. 1994), and a bootstrap
analysis (n = 100) was performed.
Maximum-parsimony (MP) analyses (Camin and Sokal 1965) were calculated with the PAUP computer program v. 3.1.1
The Phylogenetic
Status of Platyhelminthes
489
DEUTEROSTOMIA
PROTOSTOMIA +
ASCHELMINTHES”
T&lad-2
100
-&lad-l
I
Rhabdocoel
Lecithoepitheliate
Proleci thonhoran
r,
‘PLATYHELMINTHES’
I
I
’
Macrostokrid-1
Macrostomid-2
Catenulid
Gastrotrich
Acanthocephalan
ASCHELMINTHES
Acoel-2
I
Acoel-1
I
‘PLATYHELMINTHES
DIPLOBLASTS
I
I PROTOZOA
Choanoflagellate
0.05
FIG. 3.-Neighbor-joining
tree including two acoel sequences. All branch lengths are drawn to scale. Note long branches leading to and
separating the two acoel species, which indicates they may be fast-clock organisms. Lack of resolution within the Bilateria clade results from
the loss of information due to the difficulty in aligning the acoel sequences. Note that Platyhelminthes
cannot be considered monophyletic
because some Aschelminthes
branch between different Platyhelminthes
clades, here represented as “Platyhelminthes.”
Numbers at nodes are
percentages of 1,000 bootstrap replicates that support the branch. Values are shown only if over 50%. This is an unrooted tree, rooted defining
the choanoflagellate
as the outgroup. For species names, see tables 1 and 2.
(Swofford 1993) using a heuristic search procedure and
a branch-swapping
algorithm. A bootstrap analysis (n =
100) was also performed. The trees were rooted using
the SSU rRNA sequence
of the “choanoflagellate”
Sphaeroeca volvox. In all analyses, gaps were considered as missing data.
PRN (Lecointre et al. 1994) was performed using
the program MUST v. 1.0, to prepare the data, and PRN
v. 1 to test the robustness of the nodes obtained. Instead
of simply examining the bootstrap proportions
(BP) at
important nodes as a criterion of robustness of the corresponding
nodes, PRN introduces
a procedure of BP
analysis which involves following the values of BP as
a function of increasing
number of nucleotides.
PRN
was used for the same taxa used in the distance analysis,
but only the parsimony-informative
positions were included, under the following conditions. The alignments
of the N species were each submitted to random sampling of a given number of sites through the use of the
PRN program running on UNIX platforms. Ten different
sequence lengths were chosen (10, 15, 25, 50, 75, 100,
150, 200, 250, 300), and, for each, 200 samples were
drawn. Thus a total of 2,000 subsets of sequence alignments were obtained, each including all N species. Each
of these subsets was used to construct a neighbor-joining
tree, which was submitted to 1,000 bootstrap replicates.
Selection of the nodes was then carried out to keep only
those with an ascending
tendency and that appeared
more frequently. At a given node, one could therefore
graphically display the evolution of BP as a function of
the number of nucleotides used to generate the tree.
The sequence alignment and the weighting mask
which determined the nucleotide positions taken for the
analyses
are available
from
the ftp site: porthos.bio.ub.es/users/ftp/pub/teu/l8Sphylogeny.
Results
A distance tree including all the sequences is represented in figure 3. Some features are consistent with
what is known of animal evolution. As expected, the
diploblast branched before all triploblast groups. However, the most interesting feature of this tree is the long
branches leading to and separating the two acoel representatives.
In fact, acoel sequences diverge so much
from the rest of the organisms studied (not only from
Platyhelminthes)
that alignment of sequences turned out
to be very difficult. Nonetheless,
at the cost of losing
many informative
sites, the two acoels and all the representatives
from the other groups (diploblasts, protostomes, “aschelrninthes,”
deuterostomes,
and platyhelminths) were included.
The analysis considers
only
490
Carranza
et al.
Cephalochordate
DEUTEROSTOMIA
PROTOSTOMIA
Arthropod-2
Arthropod-l
1001
J
ASCHELMINTHES
Triclad-1
T&lad-2
Rhabdocoel
Proleci thonhoran
Proseriate-1
a
Proseriate-2
Lecithoenitheliate
Tremkode-1
Trematode-2
__I A
111
r-l,
1001
99 q
i
100
PLATYJTELMINTHES
Nemertodern%fode
r Polvclad-1
Catenulid
Placozoan
Cnidarian
DIPLOBLASTS
C tenophore
Choanoflagellate
PROTOZOA
FIG. 4.-Distance
tree. Fitch-Margoliash
(F-M) and neighbor-joining
(N-J) methods gave the same topology, only the second being represented here. Note that Platyhelminthes
cannot be considered monophyletic because the Catenulida form an early-branching
sister group to the
rest of Bilateria, whereas the rest of Platyhelminthes
(=Rhabditophora
+ Nemertodermatida)
constitute a clear monophyletic
group which
appears as the sister group of a protostomate
+ deuterostomate
clade. Numbers at nodes are percentages of 1,000 bootstrap replicates that
support the branch, only values over 50% being represented, with the exception of the bilaterian sister group to Catenulida (45%). All branch
lengths are drawn to scale. For species names, see tables 1 and 2.
those positions that were unambigously
aligned for the
two acoels. This reduced the available positions from
1,322 to 1,13 1 and the variables from 675 to 5 12. In
this tree (rooted with the choanoflagellate
Sphaeroeca
volvox), the acoels constitute an early sister branch to
the rest of bilaterians (67% bootstrap). However, the loss
of informative
sites leaves the internal relationships
of
the bilaterian group unresolved. The very long branches
of the two acoels suggest that we are in front of fastclock organisms; hence, their position in the tree could
be artifactual. To avoid their disturbing influence, they
were not included in subsequent analyses.
The distance analyses, Fitch-Margoliash
and neighbor-joining
methods, not including the acoels, resulted
in identical topologies (fig. 4). Two general features of
the tree were unexpected. First, Platyhelminthes
appear
to be a paraphyletic
group, as the order Catenulida do
not cluster with the rest of Platyhelminthes.
Although
weakly supported (45% bootstrap), Catenulida appears
in this tree as the most primitive bilaterian, as it is the
first branching after the diblastics. PRN analysis shows
the node for Catenulida
+ Diplobasts
+ choanoflagellate versus the rest of organisms to be a promising node
(results not shown), although almost 2,000 parsimonyinformative
sites will be needed to obtain 100% boot-
strap. Second, the rest of Platyelminthes
forms a monophyletic clade (99% bootstrap) which appears, although
only weakly supported (less than 50% bootstrap), as the
sister group of the rest of the Bilateria. However, protostomes do not form a monophyletic
group but a paraphyletic one, as nematomorphs,
acantocephalans,
and
gastrotrichs
branch inside them. Moreover, and even
more significant, the degree of divergence inside the Platyhelminthes
ranks with that among protostomes
and
deuterostomes
(compare the long branches separating
any two platyhelminths
to the branches separating protostomes or deuterostomes).
The 50% majority-rule
consensus tree of 10 maximum-parsimony
(MP) trees of equal length is shown in
figure 5. It has a length of 1,943 steps, with a consistency index (CI) of 0.495 and a retention index (RI) of
0.504. Platyhelminthes
and Deuterostomia
are well-supported monophyletic
groups (91% and 8 l%, respectively), while the Protostomia show a sequential branching
pattern not well supported by bootstrap (only 27%), similar to that obtained in the distance tree (fig. 4). Although most of the more parsimonious
trees (9 of 10)
group platyhelminths
with deuterostomes,
this grouping
is not supported by bootstrap (only 24%). This analysis
and the bootstrap analyses suggest that the available se-
1.
- -. -~ _._ ___~___ -________~
-
The Phylogenetic
c3;gate
I
I
241
60
34
801
10
100
90
86
100
54
80
71
100
91
100
100
1001
47
100
491
DEUTEROSTOMIA
d
lOO-
66
24
Status of Platyhelminthes
10
100
27
90
100
Proleci thophoran
Rhabdocoel
Leci thoepi theli ate
Proseriate-1
Proseriate-2
Nemertodermate
Trematode-1
Trematode-2
Cestode
Polyclad-1
Polyclad-2
Macrostomid-2
Macrostomid-1
Annelid-l
AnnelidMollusc-1
Nemerti ne
Mollusc-2
Arthropod-l
Arthropod-2
Catenuli d
Placozoan
Cnidarian
Ctenophore
Choanoflagellate
‘PLATYJTELMINTHES
PROTOSTOMIA
1 ‘PLATYHELMINTHES
DIPLOBLASTS
I
1 PROTOZOA
FIG. 5.-Fifty
percent majority-rule
consensus of 10 most parsimonious trees (percentiles indicated below the nodes). Bootstrap values are
indicated above the nodes, only those over 50% being shown. Total length of the tree is 1,943 steps; the overall consistency index is 0.495.
Branch lengths are unrelated to evolutionary
distance. The topology of this tree only differs from the distance tree of figure 4 in that the
monophyletic
‘Platyhelminthes’
(=Rhabditophora
+ Nemertodermatida;
Ehlers 1985) form a clade with the deuterostomes.
However, the low
bootstrap values for this clade and for the protostome lineage suggests instead a polytomy (see text). For species names, see tables 1 and 2.
quence data are insufficient to unambiguously
infer the
branching order among the three groups (protostomes,
deuterostomes,
and platyhelminthes).
The order Catenulida is, again, the sister group of the rest of bilaterians,
with a bootstrap value of 71%.
Maximum likelihood (ML; fig. 6) yielded a similar
topology to the distance tree (fig. 4) as regards the paraphyly of protostomates.
Again, pseudocoelomates
(here
represented
by the nematomorph
Gordius) fall within
the protostomate
clade branching with the two arthropods and forming a sister group to a group formed by
the monophyletic
Platyhelminthes
(excluding the Catenulida) and the other pseudocoelomates,
the acanthocephalan Moliniformis,
and the gastrotrich Lepidodermellu. In this analysis, Platyhelminthes
as well as pseudocoelomates
appear buried within a general protostomate clade. Again, the exception
is the Catenulida,
which is the first taxon branching after the diploblastics,
although the bootstrap value for the Protostome + Deuterostome + Platyhelminthes
clade is again low (45%).
The resulting internal phylogeny
of Platyhelminthes is basically the same with all the methods used
(figs. 3-6). As stated above, Catenulida do not cluster
inside the Platyhelminthes,
and it is not possible to establish the position of Acoela. The main monophyletic
group corresponds to Rhabditophora
(sensu Ehlers 1985,
1986) plus Nemertodermatida
and includes the parasitic
species (Neodermata).
This group divides, in turn, into
two monophyletic
clades: one including the orders Macrostomida
and Polycladida;
the other, the rest of the
orders including the parasitic classes. Maximum-parsimony trees, however, show an early branching of Mucrostomum (family Macrostomidae)
as compared to Microstomum
(family Microstomidae)
plus Polycladida
(fig. 5). Moreover, in the ML tree (fig. 6) we find the
two macrostomids
branching independently
within this
clade. PRN analysis shows this to be due to the fact that
Macrostomum
is more divergent from the Polycladida
than Microstomum
is (fig. 7). The node for Microstomum + Macrostomum
(fig. 7A) is better supported than
the ones joining Polycladida to either Microstomum (fig.
7B) or Mucrostomum
(fig. 7C), and nearly as well as
the one joining
Polycladida
and Macrostomida
as a
whole (fig. 70). However, the uncertain clustering of
Macrostomum
with the Polycladida, due to a greater divergence, should cause the existence of a greater number
of homoplasies of this species to other species from outer groups, and this makes it branch independently
in the
MP and ML analysis.
Leaving aside the clade formed by Macrostomida
and Polycladida, the internal relationships
of the rest of
Rhabditophora
+ Nemertodermatida
are not well resolved, with the exception of three clear monophyletic
lineages: Tricladida (Crenobia + Dendrocoelum),
Proseriata (Monocelis + Archiloa), and the parasites (Lobatostoma,
Echinococcus,
and Schistosoma),
all with
maximum bootstraps (100%).
Discussion
This paper explores one of the most important unsolved problems in systematic biology, the origin of Bi-
492
Carranza
et al.
DEUTEROSTOMIA
L-r
56
I
Mollusc-1
Annelid-l
PROTOSTOMIA
I
Nematomorph
Gastrotrich
ASCHELMINTHES
AcanthoceDhalan
Triilad-1
I
c
Triclad-2
Rhabdocoel
Leci thoepi theliate
Proleci thophoran
PLATYHELMINTHES
_jJ_
Cestode
Nemertodermate
Macrostomid-1
_gj
,-
-
Macrostomid-2
Catenulid
DIPLOBLASTS
PROTOZOA
0.05
FIG. 6.-Maximum-likelihood
tree including representatives
from Nematomorpha,
Gastrotricha, and Acantocephala.
As in the distance and
MP trees, the Catenulida is the most primitive bilaterian and protostomes cannot be considered monophyletic. The monophyletic Platyhelminthes
(=Rhabditophora
+ Nemertodermatida;
Ehlers 1985) are sister to a clade including Gastrotricha and Nematomorpha
and both are buried within
the protostomian
phyla. Numbers at nodes are percentages of 100 bootstrap replicates that support the branch, only those over 50% being
represented, with the exception of the sister group to Catenulida (45%). For species names, see tables 1 and 2.
lateria. Using complete 18s rDNA sequences and different methods of phylogenetic
analysis, we have studied the monophyletism,
phylogenetic
position, and internal phylogeny of one of the most likely candidates
for sister group of all the other Bilateria, the Platyhelminthes. The analyses show the Catenulida branching
after the diploblasts, whereas the Rhabditophora,
which
form a monophyletic
clade, branch early on in the evolution of the protostomates.
In addition, all analyses give
a similar internal phylogeny for the Rhabditophora,
with
the parasites as a monophyletic
group branching unexpectedly early within the evolution of the group.
The Acoela:
Organisms?
Primitive
or Derived
Fast-Clock
Our 18s analyses show Acoela as the first Bilateria
to branch after the diploblasts, although with only moderate support (67% bootstrap) (fig. 3). An early divergence of acoel flatworms in triploblast evolution had
previously
been reported by Katayama
et al. (1993)
based on partial 18s rDNA sequences. Later, Katayama
et al. (1995) used the acoel Convoluta naikaiensis with
two dicyemids,
one mixozoan, the nematode Caenorhabditis elegans, and other diploblast and triploblast organisms to position the mesozoan dicyemids within the
Metazoa. A clade made by the acoel, dicyemids, mix-
ozoans, and C. elegans was found to be the sister group
to the rest of Bilateria. All the members of this clade,
however, had very long branches, which may explain
why species so diverse grouped together.
The NJ tree supporting
the early branching
of
acoels (fig. 3) leads, however, to several inconsistencies.
First, protostomes
and deuterostomes
cluster together
but with low bootstrap (less than 50%), and protostomes
appear paraphyletic,
with arthropods and the single nematomorph included forming a sister group to the deuterostomes. When acoels are not introduced, deuterostomes appear highly supported (94%, 81%, and 84% in
NJ, MP and ML trees, respectively;
figs. 4-6), whereas
protostomes remain paraphyletic because aschelminthes
(NJ trees; fig. 4) or Platyhehninthes
(ML trees; fig. 6)
appear buried within them. Second, the bulk of Platyhelminthes appears only weakly supported (5 1% in fig.
3); instead, when acoels are not included, Platyhelminthes are very highly supported (figs. 4-6; see below).
Finally, and most importantly,
the lines leading to and
separating the two acoels are extremely long, which may
lead to artifactual grouping. Indeed, another long-branch
Moliniformis, is attracted
organism, the acanthocephalan
close to acoels. Early branching of acoels may be explained in two ways. Either they really are very primi-
The Phylogenetic
Status of Platyhelminthes
493
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informative
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(1
sites
number
21-1
of parsimony
321
informntive
sites
FIG. 7.-The
patterns of resolved nodes (PRN) for four nodes taken from the phylogenies of figures 4-6 concerning the clustering of
Polycladida and Macrostomida.
In each graph, the 200 bootstrap proportions (ordinate) obtained for a given number of nucleotides (10, 15, 25,
50, 75, 100, 150, 200, 250, and 300) are plotted vertically as a function of increased number of nucleotides (abscissa) (for further details, see
Materials and Methods). The graphs correspond respectively to the monophyly of (A) Macrostomida
(Microstomum and Mucrostomum),
(B)
Polycladida (Plunoceru and Discocelis) + Microsfomum, (C) Polycladida (Plunoceru and Discocelis) + Mucrostomum, and (D) Macrostomida
(Mucrostomum and Microstomum)
+ Polycladida (Plunoceru and Discocelis).
tive triploblastic
metazoans or, alternatively,
they have
or have had a higher rate of molecular
substitutions
(fast-clock organisms). Fast-clock organisms (e.g., Drosophila, Caenorhabditis
elegans and most nematodes,
Artemia salina, Aedes, among others) have previously
been described and they have the common features of
clustering
together and of branching
more deeply in
trees than they should, introducing
systematic
errors
when including
them in a phylogeny
(Olsen 1987).
Fast-clock
organisms
have been linked to short
generation
times or to rate variation among lineages
(Golding 1996). Indeed, Drosophila and C. elegans have
very short generation times. Data on reproductive rates
in acoels are scant, although they are known to have
generation times of 3-4 weeks in optimal conditions
(Faubel, personal communication).
This is considerably
shorter than most generation times reported for other
Turbellaria (Heitkamp 1988) and gives support for such
an effect in acoels. Whether their long branches stem
from shorter generation times as predicted by the generation time effect hypothesis or whether they are due
to increased rates of change in their lineage is an open
question. Acoels have been proposed in the past to be
the most primitive Platyhelminthes.
However, most features considered primitive in acoels are based on negative evidence
(absence
of characters),
though the
494
Carranza
et al.
“brain” and the longitudinal
nerve cords closely resemble the cnidarian and the ctenophore
condition (Hazsprunar
1996). Recent reappraisals,
however, place
acoels as a rather derived group (Ehlers 1985; Smith,
Tyler, and Rieger 1986; Willmer 1990).
Are the Catenulida
an Early Bilaterian
Group?
In the past, Catenulida have been considered as an
aberrant member of Platyhelminthes
(Reisinger 1924) or
to represent one of the first offshots in their evolution
(Ax 1963; Karling
1974). Some (Sterrer and Rieger
1974) have raised the question of whether this group
should even be classified within the Platyhelminthes.
This is because the postulated synapomorphies
between
Catenulida
and Rhabditophora:
protonephridia,
ciliary
rootlet system, and mode of epidermal replacement (Ehlers 1985) have not been proved to be homologous
(Smith, Tyler, and Rieger 1986), and synapomorphies
linking Catenulida
to Acoelomorpha
are not known.
Synapomorphies
of the Catenulida are the unpaired excretory system, the special organization of the cyrtocyte,
the dorsorostral position of the male copulatory organ,
and aciliary spermatozoa
(Karling 1974; Ehlers 1985).
Other features are plesiomorphies,
common to most Platyhelminthes,
and some (e.g., sparsely ciliated epidermis, monociliated
epidermal sensory receptors, lack
of frontal glands, and lack of rhabdites) are common to
lower Eumetazoa.
With the exception of trees incorporating
acoels
(fig. 3), Catenulida is the first group branching after the
diploblasts,
although with low or moderate bootstrap
support (45%, 7 1%, and 45% for NJ, MP, and ML trees,
respectively).
Moreover, the promising PRN node found
for Catenulida
+ Diploblasts
+ choanoflagellates
(Protozoa) is a good indicator of the basal position of this
group. However, their basal position in all trees could
be a consequence
of a greater rate of evolutionary
change for this group (similar to what happens with
Acoela). In trees constructed using algorithms reflecting
rate inequalities along branches such as those used here
(fig. 4), one essentially carries out a relative-rate test on
several species simultaneously,
a rate difference being
manifested by a difference in branch lengths (Philippe
et al. 1994). Clearly at variance to what happened with
acoels, the catenulid branch is similar in length to the
rest of metazoans (only some platyhelminths
have somewhat longer branches). A relative-rate test (Wilson, Carlson, and White 1977) was carried out, resulting in very
similar distances
for deuterostomes
(2 1,8%), protostomes (21,4%), “Platyhelminthes”
(23,5%), and Catenulida (Stenostomum) (20,7%) when calculated using the
choanoflagellates
as an outgroup. Similar results are obtained when the diploblasts are used as outgroup (18%
for deuterostomes,
17.9% for protostomes,
19.2% for
and 17.6% for the Catenulida with
“Platyhelminthes”
respect to the ctenophore).
In all cases, only the “Platyhehninthes”
seem to have slightly higher rates.
When “lower” groups, reportedly considered basal
to the triploblastic
Bilateria
(Winnepenninckx
et al.
1995), such as phylum Gastrotricha,
phylum Acanthocephala, and phylum Nematomorpha
(classically
clas-
sified as phylum Aschelminthes)
were included in the
analysis, they fell within the protostomates (NJ tree, fig.
4) or formed the sister group of Rhabditophora
(ML
tree, fig. 6, for Gastrotricha
and Acanthocephalan).
In
both cases, Catenulida branched earlier than any other
Bilateria,
including
these presumptive
lower groups.
However, when acoels are introduced (NJ trees, fig. 3),
acantocephalans
and gastrotricha branch sequentially after them, although this probably results from attraction
among long-branch
groups. Gastrotricha
and Acanthocephala were reported by Winnepenninckx
et al. (1995)
to form a weakly supported clade with the Platyhelminthes. Our analyses, using more species, support the relationship of rhabditophoran
platyhelminthes
with these
aschelminth groups, calling for further studies.
Altogether, and despite the fact that only a single
representative
has been sequenced so far, our data support the hypothesis that Catenulida constitute an independent clade that branched off early in the evolution
of Bilateria and, hence, that Platyhelminthes
probably
are paraphyletic.
The Bulk of “Platyhelminthes”:
Rhabditophora
the Monophyletic
The bulk of “Platyhelminthes,”
the so-called
sensu Ehlers (1985), appear in all analRhabditophora
yses as a clear monophyletic
group. Rhabditophora,
which includes the parasitic classes (Neodermata),
has
a main autapomorphy,
the presence of lamellated rhabdites, and several synapomorphies
such as the presence
of duo-gland
adhesive system and duo-cell weir and
multiciliated
terminal cell in the protonephridia.
There
is ample consensus in considering the Rhabditophora
as
monophyletic
(Karling 1974; Ehlers 1985; Smith, Tyler,
and Rieger 1986; Rohde 1990). This is supported here
by very high or moderate bootstraps (99% and 91% for
NJ and MP trees, respectively,
and 65% for ML trees)
and agrees with the morphological
character-based
phylogenetic schemes of Karling and Ehlers (see fig. 2). 18s
sequence data, however, show a major difference with
them: the clustering of Nemertodermatida
within Rhabditophora and not with Acoela forming the Acoelomorpha sensu Ehlers (1985). Because only one species
(Nemertinoides elongatus) has been analyzed here, the
position of nemertodermatids
should be left open.
Internally,
NJ and ML trees reproduce two main
monophyletic
clades with high or moderate bootstrap
values: Macrostomida
+ Polycladida
and the rest of
Rhabditophora
plus Nemertodermatida.
Macrostomida
and Polycladida
appear as close groups in Karling’s
( 1974) and Ehlers’ ( 1985) phylogenetic
proposals (fig.
2) but do not cluster together. Smith, Tyler, and Rieger
(1986) see Polycladida within a clade with the bulk of
Rhabditophora,
whereas Macrostomida
form a sister
group to the enigmatic Haplopharyngida.
Finally, according to Rohde (1990), Macrostomida
form a clade
with Prolecithophora
this being the sister group of Proseriata and Neodermata.
This state of flux as regards
Macrostomida
may reflect the uncertainties
of linking
Macrostomidae
and Microstomidae
in a single order
(Rieger, personal communication),
indicating the need
The Phylogenetic
to include more species in future analyses. Despite these
difficulties,
the high bootstraps found in NJ trees, the
promising PRN nodes for Microstomidae
+ Macrostomidae (fig. 7A) and Polycladida
+ Macrostomidae
(fig.
7D), and the presence of homocellular
gonads and endolecithal eggs in both groups are good indicators of
close affinities between Polycladida and Macrostomida.
The sister group to Polycladida
+ Macrostomida
reproduces the Neoophora (Westblad 1948; Ehlers 1985)
with high or moderate bootstraps (78%, 86%, and 57%
for NJ, MP and ML trees, respectively).
The main synapomorphies
of this group are the presence of heterocellular female gonads and the ectolecithal
eggs. The
internal phylogeny of Neoophora, however, is not well
resolved. Rohde (1990) has noted that apomorphies for
the Lecithoepitheliata,
Prolecithophora,
Proseriata, and
Tricladida are insufficient
to determine
their position
within the Platyhelminthes.
The low bootstraps found
here for most neoophoran branchings indicate that at the
present level of knowledge,
18s rDNA sequences are
unable to settle the issue. Even so, all trees reproduce
some interesting regularities. First, leaving Nemertodermatida aside, parasites appear as a clear monophyletic
group basal to the rest of neoophorans (see below). Second, Tricladida often clusters with Rhabdocoela
with
low or moderate bootstraps (although PRN nodes are
promising;
data not shown), forming a highly derived
group. And finally, Proseriata always show a rather basal
position close to the parasites (Neodermata)
and far
from their presumed sister group, the Tiicladida. These
results indicate that Seriata are a paraphyletic group and
that Neodermata are a monophyletic
basal group.
Parasitic
platyhelminthes
(Neodermata;
Ehlers
1985) are most often considered either the sister group
of the Dalyellioida,
rhabdocoels with doliiform pharynx
(Ehlers 1985; Brooks 1989), or an early branch of rhabdocoels which retained the less specialized
rosulate
pharynx (Kotikova and Joffe 1988). Based on the similarity between ultrastructure
of the flame bulbs of Neodermata and those of most “primitive”
proseriates, Rohde (1990) considers them not related to rhabdocoels but
as the sister group of the Proseriata. The NJ, MP and
ML trees built here support the monophyly
of Neodermata and its basal position within the evolution of neoophorans. Cestodes begin their life cycle as parasites of
arthropods, the digenetic trematodes as parasites of mollusts. It is widely accepted that most invertebrate phyla
were established as distinct and disparate groups by the
Cambrian, as shown by the Ediacaran and Burgess Shale
fauna (Conway Morris 1993; Valentine, Erwin, and Jablonski 1996), and there are sound reasons to believe
that parasitism is as old as predation, symbiosis, and
mimicry. Therefore, it is also sound to think that certain
primitive-looking
free-living
“turbellarians”
became
parasites of invertebrates,
probably
molluscs (Rohde
1995), during Cambrian times. Later, when vertebrates
appeared, they added a fish host and a final piscivorous
host, giving the typical life cycle with two larval stages
and three host species (Stunkard 1975). This plausible
scenario perfectly fits the early branching of parasitic
groups from primitive
“turbellarians”
found here and
Status of Platyhelminthes
495
argues against proposals on parasitism as a late development from advanced turbellarian rhabdocoels (Ehlers
1985; Brooks 1989).
Taxonomic
and Phylogenetic
Considerations
The general topology of the trees here obtained
agrees to a large extent with previous proposed phylogenies of the animal kingdom (Field et al. 1988; see the
reanalysis
of their data by Patterson
1989 and Lake
1990), followed by those of Christen et al. (1991),
Adoutte and Philippe (1993), Chenuil(1993),
and Wainright et al. (1993). All show two main features: first, a
clear split between diploblasts and triploblasts; second,
a poor resolution for the major coelomate phyla, especially for protostomes often giving big multifurcations
that include annelids, molluscs, arthropods, nemertines,
and other minor phyla. In addition, the positions of
pseudocoelomates
and acoelomates
remain uncertain.
Philippe, Chenuil, and Adoutte (1994) have provided
convincing
evidence that unresolved bushy multifurcations arise because the resolving power of the presently
available complete 18s rDNA database is only about 40
Myr. There is now amply documented evidence, namely
from fossil records, that the period of early and rapid
origin and diversification
of the major invertebrate phyla, the so-called “Cambrian explosion” occurred in less
than 40 Myr. In other words, multifurcations
derived
from molecular data have provided molecular corroboration for the Cambrian “explosion.”
In this context, the
uncertain position of the Rhabditophora
as regards protostomates and deuterostomates
(see figs. 3-6) may indicate that the bulk of “Platyhelminthes”
appeared and
evolved during this fast Cambrian radiation. In contrast,
the early branching of Catenulida supports an acoelomate grade of organization
for the first bilaterian, although the small number of species studied leaves the
issue unresolved and calls for a deeper study.
As regards the hypothesis put forward on the origin
of Bilateria (fig. lA--C) the phylogenetic
derivations of
our results could also be of interest. First, Platyhelminthes as a whole cannot be considered the sister group of
the rest of Bilateria as they are paraphyletic. Second, the
bulk of “Platyhelminthes,”
represented
by the monophyletic Rhabditophora,
are more related, as could be
expected, to prostostomes
than to deuterostomes.
This
suggests that Rhabditophora
are either protostomates or,
most likely, an early branch on the lineage leading to
the bulk of protostomates
(fig. 1B). This rules out the
hypothesis that sees “Platyhelminthes”
(Rhabditophora)
as the most primitive Bilateria (fig. IA) or as a derived
group from coelomate
ancestors (fig. lc). Third, all
analyses and trees, with the exception of those incorStenostomum
porating
acoels,
show the catenulid
branching after the diploblasts and earlier than any triploblast
here studied, including
potential
primitive
groups such as gastrotrichs, acanthocephalans,
and nematomorphs (Winnepenninckx
et al. 1995).
To summarize, we have shown that Platyhelminthes
as a whole do not seem to be primitive. Although our
results do not contradict the idea of Platyhelminthes
being monophyletic,
the persistence
of the node, albeit
496
Carranza et al.
with a low bootstrap,
ter group to the rest
on its monophyly.
paucity of sampling
for a large sampling
hold promise to be
lateria.
locating the Catenulida as the sisof the Bilateria, casts some doubts
Long-branching
of acoels and the
in both acoels and catenulids calls
of these primitive groups which
the key to the evolution of the Bi-
Acknowledgments
We thank the following persons for help in locating
and providing turbellarian
material: M. Curini-Galleti,
A. Falleni, J. Gamo, B. I. Joffe, D. T J. Littlewood, M.
Litvaitis, C. Norena-Janssen,
C. Novell, R. Ponce de
Leon, M. Reuter, R. Rieger, E. Schockaert, J. Smith III,
and S. Tyler. We also thank Gonzalo Giribet, Miguel
Angel Arnedo, and Carles Ribera for helpful comments
and discussions. We are grateful to one reviewer, whose
constructive
criticism and comments
helped to refine
this manuscript.
This study was supported by a DGICYT grant PB90-0477 and a CIRIT grant GRQ93-1044
to J.B.
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MANOLO GOUY, reviewing
Accepted
January
24, 1997
editor