The interrelationships of all major groups of
Platyhelminthes: phylogenetic evidence fkom
morphology and molecules
D. T. J. LITTLEWOOD','* FLS, K. ROHDE' AND K. A. CLOUGH'
I Department of<oology. 7he Natural Hirtov A4useum, Cromwell Road. London S W 7 5BD;
'Diviiion ofL$ Sciences. King3 College London, Campden Hill Road. London W8 7 A H ;
'Department of <oology, UniversiJy of New England, Armidale. NSW 2351, Australia
We used a data matrix of 65 morphological characters from 25 ingroup and 6 outgroup
taxa, and an alignmcnt comprising complete 18s rDNA scqucnccs from 82 species of
parasitic and free-living Platylielminthcs and from 19 species of loivcr invcrtchratcs to analyse
phylogenctic relationships ofvarious platyhclminth taxa. Of the 1358 unambiguously aligiiablc
molecular positions, 995 wcrc variable and 757 \vcrc phylogcnetically informative (parsimony
criterion); complete 18s rDNA sequences ranged in length from 1755 to 2873 lip. Main
conclusions arc: Ncodermata are monophyletic, and the Trematoda, hlonogcnca and Cestoda
within thcm arc monophylctic as well. The sister group of thc Ncodcrmata is all the otlicr
Ncoophora; the Kalyptorhynchia, 'I'yphloplanida, Dalyclliida and Tcmiiocephalida form
one clade, and the last three another. Rlonophyly of the Seriata is rejected, but Polycladida/
Rfacrostomida/Haplopharyngida arc monophylctic, as arc the last two taxa. As a consequence,
validity of the taxon Trepaxonemata is rejected. Further studies must show the corrcct
position of the Acoela and Ncmcrtodermatida. It is strcsscd that morphological and molecular
data in some respects lead to contradictory results, for instance concerning the position of
the Fccampiidac/ 1~ r a . r t o n i n / Z ~ i i t / ~and
~ o ~the
h ~ ~rclativc
~
position of the Lccithoepitlicliata.
Denser sampling of taxa for molecular data, complcmcntary scqucnccs from independent
gcncs, and inclusion of additional morphological data arc necessary to resolve thcsc contradictions.
0 1:)w 'l'l1r l . i l l 1 l ~ ~Socict)
~~l
01 1.011d011
ADDITIONAL KEY WORDS:-total
c\idcnce
1% rDNA
-
~
systematics
~
cladistics.
CONIENIS
Introduction . . . . . . . . .
'Background and aims . . . .
hf~)rpholo~gy. . . . . . .
hIaterial and methods
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Choice of morphological characters
Molecular data . . . . . .
DNA extraction, gene amplification
Sequence alignmrnt . . . . .
* Corresponding
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and sequencing .
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author: E-mail:[email protected]
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Phylogenrtic analysis .
Results and discussion
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blorpholokq . . . .
nlolecules . . . . .
Combined morphological
Acknowledgements
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References . . . . . .
Appcridix 1 . . . . . .
Appendix 2 . . . . . .
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A resolved, robust working phylogeny of the Metazoa and its constituent phyla
has been, and largely still remains, a holy grail for zoologists. We require such an
insight into evolutionary history and past cladogcnetic events, not only as a basis
for systematics but as a foundation for interpreting comparative biological data in
terms of evolutionary patterns and processes. Of the 34 nominal metazoan phyla,
it is the Platyhelminthes, or a taxon including the Platyhelminthes and the Gnathostomulida (the Plathelminthomorpha), that have been widely regarded as the
earliest divergent bilaterian group and sister-group to all other triploblasts (e.g.
Willmer, 1990; Ax, 1996). In particular, the acoelomate flatworms, which are
anatomically very simple, have been implicated as the most ‘primitive’ of metazoans
largely on the basis of their lack of characters, although their status as ‘kin<pinsof
metazoan evolution’ has been argued against in the light of certain ultrastructural
features (Smith & Tyler, 1985a). Nevertheless, whether in a cladistic or phenetic
framework, and from both morphological and molecular perspectives, flatworms
are seen to occupy a pivotal position in early metazoan evolution (e.g. Willmer,
1990; Nielsen, Scharff & Eibye-Jacobsen, 1996; Shubin, Tabin & Carroll, 1997;
Van dc Peer & De Wachter, 1997).
The phylum also has attracted attention because it includes perhaps the largest
clade of obligate parasites, the Neodermata, incorporating the monogeneans, cestodes, digeneans and aspidogastreans. Neodermatans, characterized by their ‘new
skin’ which replaces thc plesiomorphous ciliated epidermis on transition from larva
to adult, parasitize an enormous number of invertebrate and vertebrate hosts (Ehlers,
1985a; Rohde, 1996) and include some of the most medically and economically
important parasites known (e.g. Schmidt & Roberts, 1996). Many of the parasitic
flatworms exhibit high host-specificity and, in turn, this has led to high estimates of
species diversity in some groups (e.g. parasites of inshore reef fishes; (Rohde, 1976;
Cribb et al., 1994)).The phylum as a whole shows a great diversity in morphology,
habitat, biogeography and life-history strategies. Furthermore, as the origins of the
neodermis and subsequently parasitism appear to have been a single, critical
evolutionary event, the group lends itself to an investigation of parasitism as
a general biological phenomenon. Brooks & McLennan (1993a,b,c) have used
comparative parasitological information from the phylum (e.g. morphological features, life-history strategies, and biogeography) to highlight the value of parasite
phylogenies in understanding general trends in evolution and adaptive radiations.
Of course, such treatises rely heavily on accurate estimates of phylogeny and the
recognition of sister-taxa. Without these it is difficult to map the evolution of
characters, to recognize them as either homologous or homoplasious and to interpret
the evolutionary radiation of the group in question. Here we present a reassessment
of available evidence, both old and new, and complement morphological evidence
with molecular data from a wide range of free-living and parasitic taxa.
Background and aims
Untangling the interrelationships of the Platyhelminthes has attracted attention
from both phylogeneticists restricting their attention to morphological characters
(e.g. Ehlers, 1984, 1985a; Brooks, O’Grady & Glen, 1985; Rohde, 1990) and those
relying solely on molecular data (Baverstock et al., 1991; Blair, 1993; Rohdc et al.,
1993;Joffe et al., 1995; Katayama, Nishioka & Yamamoto, 1996; and most recently
Carranza, Baguiia & Riutort, 1997). Most notable morphologically based scenarios
are those proposed by Ax (1984) and Ehlers (1985a) which were the first cladistic
treatments and have remained the most widely cited in spite of supplementary and
often contradictory morphological evidence that has since appeared in the literaturc
(e.g. Rohde, 1990, 199 1). Few studies have attempted to combine morphological
and DNA evidence (Rohde et al., 1995).
Recently a number of authors have utilized partial and complete 18s rRNA
sequences to resolve flatworm relationships (Baverstock et al., 1991; Blair, 1993;
Rohde et al., 1993). This switch to molecular systematics reflects the apparent
instability of the morphologically based scenarios and the problem that few morphological features are available for phylogenetic reconstruction (Rohde, 1990). The
strength of molecular data is their ability to provide independent estimates of
phylogeny. However, we feel these molecular and morphological studies are incomplete when presented in isolation. Not only has valuable phylogcnetic information
been ignored by studies concentrating exclusively on molecular or morphological
data sets, but molecular studies have relied on sparsely sampled sets of arguably
inappropriate species (exemplars) and morphological studies have been analysed
cladistically ‘by hand’, rather than using more powerful computer-based algorithms
which allow us to examine the utility of seemingly homoplasious features. Here we
aim to redress this problem by presenting new gene sequence data for 37 taxa, and
complementing complete 18s rRNA gene data previously published for 45 taxa, thus
representing each of the nominal orders of Platyhelminthes. The ‘Prolecithophora’are
represented by Urastoma and Ichthyophaga, included by Cannon (1986) in this order
but unlikely to belong to it. Subsequently, we treat them as separate taxa of uncertain
affiliations. DNA data for genuine prolecithophorans are not available, but we have
morphological information and the Prolecithophora are therefore included in our
analysis. Also, we present new and revised morphological characters and analyze
each data set rigorously, employing current phylogenetic reconstruction methods,
and testing for congruence and conflict between the morphological and molecular
data. We do not expect to solve all the problems of platyhelminth phylogenetics in
this paper, rather, we wish to consolidate morphological and molecular data, both
old and new, in a total-evidence approach so that we may reveal robust phylogenetic
relationships and indicate weaknesses in both the morphological and the molecular
data sets. In this way we make character matrices available for further scrutiny,
expansion and assessment and ultimately hope to provide a basis for a working
phylogeny for this species-rich, diverse and important group utilizing all available
systematic data.
7H
1)
1 .J 1,l I 1 l,l,\\OOl) I 7 If
In placing our data sets in contcxt it is important to review the phylogcnetic data
currently available. Here we present a brief synopsis of the key morphologically
based studies with a brief discussion of the characters uscd. We are aware of other
morphological data sets being accumulated that are attempting to address the
problcm of platyhelminth phylogcnctics, but as these are not currently available or
are incomplete we mention them simply to draw the readers’ attention. The review
of molecular studies highlights thc pitfalls associated with sampling density in terms
of species number and the size of gene fragments sequenced, as well as indicating
preliminary rcsults that have guided our study and subsequent analyses. Key issues
that have attracted a reevaluation of morphological and molecular data include
notions that Platyhelminthes arc not basal bilaterians and they are not monophyletic.
A5 a necessity, these points arc raised in thc contcxt of our own species sampling,
choice of outgroup and method of analysis.
As with any group of taxa, morphological characters of phylogenetic utility have
been accumulated by a large number of individuals. However, few have consolidated
such characters into a cladistic framework for the Platyhelminthes. Ax (1984) and
Ehlcrs (1984, 1985a, h) can claim to he the only workers to do this so far and so
cxtensivcly. Their schemes, which largely agree with onc another, remain as our
guide to putative synapomorphic features that distinguish a strictly bifurcating tree
uniting the major flatworm groups (see Fig. 1). Both these workcrs rcgard thc
Platyhelminthes as monophylctic, and the ‘turbcllarians’ as a paraphylctic group
including the largely frcc-living flatworms. Ehlers’ (1 985a,h) system is illustrated in
Figure 1. It is strictly hicrarchical. The Catenulida is the sister group of all other
Platyhelminthes (the Euplathclminthes), the Accolomorpha (Acoela plus Nemcrtodcrmatida) is the sister group of all the others except the Catenulida (Rhabditophora).
The Macrostomida is the sister group of the rest (the Trepaxonemata) beginning
with the Polycladida, etc. Importantly, all the large parasitic taxa (the Ncodermata)
are monophyletic at tlic tip of the hierarchy; i.e. they arc the most derived. Among
them, the Trematoda (Aspidogastrea plus Digenca) arc the sister group of all tlic
others. Monogcnca arc considered to be monophylctic within the Ncodermata. Both
Ax and Ehlcrs rcgard the Gnathostomulida as thc sister group of thc Platyhclminthcs,
both taxa constituting the Plathelminthomorpha (for recent information about
gnathostnmulid phylogcny see Sterrer, Mainitz & Riegcr, 1985; Riegcr & Tyler,
1995; Ahlrichs, 1997; Hcrlyn & Elilers, 1997; Littlcwood et al., 1998).
Ehlerc’ system of the PlaphelmintheJ
Ehlcrs’ (1985a; see Fig. 1) system is by far the most detailed and valuablc one.
We therefore discuss it in greater detail as a basis for a comparison with later studies
and our own conclusions. His synapomorphies and autapomorphies (all
‘autapomorphics’ in Ehlcrs’ terminoloLgy)are presented in Appendix 1 and Figure
1. We cannot critically examine all the characters in dctail uscd by Ehlers, hut some
have to he discarded in view of rccent studies or because they are unlikely t o
be homologous. For example, all of the supposed apomorphies for the Plathelminthomorpha arc found in many unrelated taxa and unlikely to he homologous
OUTGROUP
1
+
i
J
Catenulida
-pJ
rq
Nemertoderrnatida
LTJ
Acoela
Macrostomida
Polycladida
Lecithoepitheliata
Prolecithophora
-
2 Euplathelminthes
3 Acoelomorpha
6 Rhabditophora
8 Trepaxonemata
10 Neoophora
12 N.N.l
14 N.N.2
15 Seriata
18 Rhabdocoela
19 Doliopharyngiophora
20 Neoderrnata
21 Trematoda
24 Cercorneromorphae
26 Cestoda
28 Nephroposticophora
30 Cestoidea
Proseriata
Tricladida
"Typhloplanoida"
U
I
"Dalyellioida"
Aspidogastrea
Digenea
Monogenea
Gyrocotylidea
Arnphilinidea
Figure 1. The phylogeny of thc major clades of Pl;~t)-Iiclmiiitliesaccording to Ehlrrs (1!)85a,l~).T h r mnjor s)-napomorphirs are markrd
detailed in Apprndis 1. For full details. scc thc original references.
011 the
I)ranchcs and are
-s
g
130
1) 1 J 1.1 1 I l ~ l , I ~ O O
hT,II
l~
(hermaphroditism, intcrnal fertilization etc.). The same applies to the characters of
the Euplathelminthes. Various pharynx types arc used repeatedly, but, as pointed
out by Rohdc (1 990) very few ultrastructural studies of pharynges have been made
(see also below). Loss of duo-gland adhesive organs is used several times but has
little phylogcnetic value (see below). The synapomorphies for the Seriata, a particular
type of pharynx and ‘follicular gonads’, are not convincing. Cranial convergence of
rootlcts of epidermal cilia, suggested to be synapomorphic for the Proscriata by
Schockaert (cit. Ehlcrs 1985a: 41), has since also been found in a macrostomid
(Rohde, Watson & Chisholm, 1998), although the possibility cannot bc ruled out
that it has independently evolved in the two groups. Therc is no convincing
synapomorphy for the Rhabdocoela and Doliopharyngiophora (pharynx and 105s of
duo-gland adhesive organ!). Even the ‘hicrarchy’ within thc Neodermata is to a
large dcgree based on thc rcduction or loss of hooks, and it is uncertain where
incorporation of a vcrtcbrate host in thc life cycle has occurred.
Choice
of morphological characters
Critical for any phylogcnetic analysis is a proper distinction of homologous from
non-homologous characters. Pattcrson (1982), Rieger and Tyler (1 985) and various
authors in Hall (1 994)’ among others, have given recent discussions of homolo<gy.
Rohde (1 996) has shown that the use of characters in phylogcnetic analyses which,
prior to the analysis, have not bccn examined critically for their homoloLgy,may
lead to wrong conclusions. This is particularly true for groups, such as parasitcs,
that havc been exposed to common selection pressures. The use of a few charactcrs
whosc homology has been madc likely by careful assessment is more likely to reveal
phylogenetic relationships than analyses using large numbers of unassessed characters.
In general, loss of characters or reductions have to be particularly carefully assessed
for homology, bccausc such losses or rcductions have occurred frcqucntly and in
many evolutionary lines. They may ncvcrthcless be useful after assessment. Thus,
for instance, Ehlers (1985a) has shown that the lack of a mitochondrion in mature
sperm is a synapomorphy of the Cestoidea (see also Justinc, 1991, 1995). Also, in
general, the usefulness of characters rises with their complexity, bccause complex
organs in two or more taxa that correspond in all or most of their components with
cach other, are unlikely to havc cvolvcd independently more than once. Such
correspondence or organs can be shown in much grcater detail by electronmicroscopic than by light-microscopic studies. Hence, complcx organs or tissues
examined at the ultrastructural level must be given particularly great weight.
Applied to the Platyhelminthes, studies by Rohdc (1 990, 1991, further references
therein) have shown that the ultrastructure of protonephridia contains much phylogenetic information. Rohdc (1 990) has made a homology analysis of platyhelminth
flame bulbs applying the homoloLgycriteria of Remane (1952) and othcrs and
summarizcd by Ricgcr and Tyler (1985), as well as functional criteria of Rieger and
Tyler (1985), and concluded (p.986) that thc homoloLgyof the terminal parts (flamc
bulbs) of thc protoncphridia between various taxa of Platyhelminthcs is very likely,
and that great weight must be given to it in cstablishing phylogenetic systems. Other
characters likely to be useful (and used by various authors, e.g. Ehlcrs, 1985a) arc,
according to Rohde (1 990), replacement of lar\ral epidermis by a tegument (neodermis), structurc of epidermal cilia, presence of electron-dense collars of sensory
receptors, sperm structure and spermiogcnesis, etc. O n the other hand, pharynx
structure, used prominently by Ax and Ehlers, was shown to be of little usc at least
for the ‘Doliopharyngiophora’ by Joffe (1987),Joffe, Slusarev & Timofecva (1987),
Joffc & Chubrik (1988) (see discussion in Rhode, 1990: 982-984). The same applies
to thc posterior attachment organ (‘sucker’)of various Ncodermata, prominently used
by Brooks and McLennan (1 993a-c), because suckers of Udonellu, temnocephalids,
monogeneans and digencans arc unlikely to be homologous (Rohde, 1990: 984;
Rohde & Watson, 1995).
Somc of the characters used in our analysis were intrcpreted differently by Ehlcrs
(1 985a). F2‘c use the Cestoidea to makc the point. Ehlcrs considered an “extremely
leaf-like structurc” as autapomorphic for the Amphilinidea, but it is also found in
the Gyrocotylidea, although they have a (sometimes very small) rosette, which
modifies the body shapc somewhat, apparcntly an adaptation to life in the digestive
tract; we therefore use this character as a synapomorphy for the Gyrocotylidea plus
Amphilinidea. He further considers 10 hooks as an ‘autapomorphy’ of thc Cestoda,
and six hooks as an ‘autapomorphy’ of the Cestoidea. However, several other
possibilities exist: 10 hooks may be due to secondary addition to the original six, or
both the Cestoidca and the Amphilinidea plus Gyrocotylidea have independently
evolvcd from a taxon with a larger number of hooks, c.g. 16 as found in many
monogeneam (some of which have tcn hooks). In other words, six hooks may be a
synapomorphy of the first and 10 hooks may be a synapomorphy of the second
group. On balancc, we consider it more likely that both body shapc and number
of hooks are synapomorphics of the two taxa Gyrocotylidca/Amphilinidea and
Cestoidea, respectivcly, as used in our data matrix, and not ‘autapomorphics’ in the
sense of Ehlers.
A large number of clectron-microscopic studies on sperm and spcrmiogenesis of
platyhelminths have been made (reviews by B2 & Marchand, 1995 on cestodes,
Justine, 1995 on parasitic flatworms; Watson & Rohde, 1995 on turbellarians);
Watson and Rohde have compiled the information available for the turbellarians
in a data matrix. However, it must be crnphasized that this matrix cannot be used
for a cladistic analysis, because homology analyses for the various characters have
not been made as yet. Very few of the characters in the matrix arc likely to be
useful, and those that are, are included in our matrix.
In our data matrix, we use only characters that arc likely to be homologous across
taxa, although we realize that any homolo<gyassumption is hypothetical and must
be open to some doubt. \+‘here there is a high degree of uncertainty, we use 0/1,
i.e. we assume that a certain ‘synapomorphy’ may either be valid or not. We include
only synapomorphics useful in a phylogenetic analysis, but not autapomorphics of
terminal taxa that do not contribute to establishing trees. Some characters are given
a ‘1’ in our matrix even if they are absent in certain taxa, if loss is likely to be
secondary. We include characters even if they arc not found in all or most species
of a larger taxon, if they are likely to be homologous with such characters in other
taxa (e.g. 38 for thc Prolecithophora).
Synapomorphies in our data matrix are based on the following references:
Ivanov, 1952; Karling, 1974; Riegcr & Tyler, 1974; Ricger, 1976; Tyler, 1976;
:12
Ehlers, 1977; Riegcr & Ricger, 1977; ‘I’cuchert, 1977; Tyler, 1976, 1977, 1979;
Tyler & Ricger, 1977; Tyler, Mclanson & Ricger, 1980; l’yler & Reiger, 1980;
Smith et al., 1982; Tyler, 1984; Ehlcrs, 1985a,b; Kornakova, 1985; Smith &
‘l’yler, 1985a,b; Bruggcmann, 1986; Smith & ‘Tyler, 1986; Smith, Tyler & Rieger,
1986; Thomas, 1986; Xylandcr, 1986; Joffe, 1987; Joffe, Slusarev & Timofeeva,
1987; Joffe & Chubrik, 1988; Xylandcr, 1988; Riser, 1989; Rohde, Watson &
Roubal, 1989; Rohde, 1990; .Justine, 1991; Rieger et al., 1991; Rohde, 1991;
Schram, 1991; \Vatson, Steincr & Rohde, 1991; Ehlcrs, 1992a,b; Rohde &
Watson, 1992; Rohde, Watson & Roubal, 1992; Rohdc, Watson & Jondelius,
1992; Watson, Rohdc & Lanfranclii, 1992; Auladcll, Garcia & Bagufia, 1993;
Ehlers & Sopott-Ehlers, 1993; Rohde & Watson, 1993; Watson & Rohde, 1993a,
h; Rohdc, 1994; Rohde & Watson, 1994a,b; BB & Marchand, 1995; Ehlcrs,
1995; Justinc, 1995; Niclsen, 1995; Ricger C(r Tyler, 1995; Rohde et al., 1995;
Rohdc & Watson, 1995; Watson & Rohde, 1995; Haszprunar 1996a,b; Lundin
& Hendelbcrg, 1996; Nielscn, ScharfT & Eibyc-Jacohsen, 1996; Rohde, 1996;
Wallace, Ricci & Melonc, 1996; Ehlers & Sopott-Ehlcrs, 1997b,c; Lundin, 1997;
Watson, 1997; Rohdc & Faubel, submitted.
Note that these references include some that discuss the valuc of synapomorphics
cstablishcd by othcrs and included in our matrix, or argue against homology of
ccrtairi characters which were therefore not included in the matrix. We hare uscd
strictly binary coding for two reasons. Firstly, binary coding is vastly superior to
multistatc coding in our initial assessment because it allows an easy ‘grasp’ of
important characters. For example, all the different types of flame bulbs of protoiiephridia could he treatcd as a single multistate character, but such a character
would be vcry confusing. Secondly, multistate coding implies homolo,gy of thc
character thus treated. For example, treating the ‘suckers’ of various Neodcrmata
as a multistatc character would make the totally unjustified assumption that different
types of sucker are indeed in somc way homologous, hut they arc really entirely
different structures (Rohdc et al., 1995). In the future, refinements of our matrix
may warrant recoding and the rcevaluation of certain characters.
We cxpect that future studies will use additional synapomorphies, somc of
which were not included in our analysis becausc we consider the available
information as iiot yet sufficicnt, or because usefulncss as synapomorphies has
been demonstrated only for smaller taxa, not included in our analysis. Almost
certainly, ultrastructural studies of pharynges will contribute significantly, as will
furthcr studies of the eye contributing to the already impressive body of kriowlcdgc
built up by Sopott-Ehlers (c.g. 1984, 1990, 199la, 1992a, 1993a,b, 1995a,h,c,
1996), Kuriert & Ehlcrs (1987), and Watson (1998), ctc. Likewise, clcctronniicroscopic studies of oocytes and vitellocytes and gonads in geiieral will be
useful (see c.g. Grcmigni, 1979, 1992; Sopott-Ehlers, 1990, 199111, 1992b, 1997;
Grcmigni & Falleni, 1992; Falleni & Grcmigni, 1993; and others). Concerning
the eyes, for instance, lenses dcrivcd from mitochondria havr been found in a
number o f free-living and parasitic flatworms, but iiot yet in the hfacrostomida,
Polycladida, Tricladida, Prolecithophora or Proseriata. Their occurrence in some
taxa may indicate common ancestry, or they may be indcpcndcntly derived
(Watson, 1998), the second alternative not unlikcly in view of the great adaptive
\.slue o f effective eyes and thc differences in dctails of cyc and lens structure
Iietwccn taxa.
12101ecular data
We chose to complement our morphological data set with complete gene sequences
of the small subunit 18s rKNA gene (rDNA) on the basis that this gene has wide
phylogcnctic utility at many levels; regions of relatively high sequence variability
are framed by regions of high sequence conscrvation, allowing easy alignment and
the establishment of base-position homoloLgybetween taxa (e.g. Hascgawa et al.,
1985; Hillis et al., 1996: 337). Furthermore, the availability of 45 ingroup and 19
suitable outgroup 18s rRNA sequences on EhlBIJGcnBank set a precedent to
provide a phylum-wide survey of this gene. T h r gene sequences utilised, and their
source, is listed in Table 1.
DuW Prtrartion, gme arn,blifiration and requeming
Specimens were fixed and stored in a minimum of five volumes of 95-100"/0
ethanol. Prior to DNA extraction, individual worms, or pieces of larger flatworms,
were rehydrated in two washes and one 1 h soak in T E (pH 8.0). Individuals were
ground in 150 pl T E (pH 8.0), 0.So/0 SDS, and digested for 3 4 h with the addition
of 6 p1 protcinase K (10 mg/ml) at 37°C. Genomic DNA was phcnol-chloroform
extracted and precipitated over 15 min at - 20°C in the presence of 0.1 vol sodium
acetate, pH 4.5-6.0, and 2.5 vols 100"/0 ethanol. After washing in 70% ethanol
DNA pellets were dried and redissolved in T E (pH 8.0).
Complete 18s rDNA was amplified from each extract with PCR (Saiki et al.,
1988) using the following primers A and B; 5'-AnlCTGGTTGATCCTGCCA~
and 5'-AGGTGAACCTGCAGAIGGATCA respectikely . Standard 50 pl PCR
reactions were set up (final concentrations: 200 phl each dNTP, 2 mhl MgCl,,
1 x reaction buffer (Perkin-Elmer), 1 U 7aq polymerase (Amplitaq, Perkin Elmer)
and cycling conditions were: hot start (95"C/5 min) followed by 30 cycles of94"@/
1 min, 50"C/1 min and 72"C/1 min. Succcssful primary amplification was achieved
with most templates but a secondary ncsted-PCR was required to amplify the gene
from some taxa. The nested-PCR involved a secondary amplification using the
purified (Wizard, Promega) products of the primary PCR reaction ( 1 p1 of primary
reaction) in two subsequent PCR reactions; onc tube contained the 5'-primer A
plus an internal 3'-primer, and another tuhc contained an internal 5'-primcr and
the 3'-primer B. The amplified products overlapped onc another by approximately
1000 bp. At least two reactions were perfbrmed for each template. Amplified products
were run on a 1O/O TAE agarose gel, cut out, pooled and purified with IYizard Preps
(Promega).
Gene fragments were directly sequenced using standard reaction mixes and
procedures on a 373 ABI automated scqucnccr with the PRIShl dye terminator
cycle sequencing ready reaction kit (ABI, Perkin Elmer). The 18s rDNA fragmrnt
was sequenced using primers A and B and between 1 1 and 13 other standard
(cukaryote-specific)internal primers. Both strands of the DNA were sequenced and
contigs were assembled with Sequencher v.3.0 (Gene Code5 Corporation, MI).
Full 18s rRNA gene sequences have been deposited with EhlBL/GenBank undcr
accession numbers detailed in Table 1. The table also indicates the identity of a
range of outgroup taxa used to root the tree.
114
1). '1'. ,J. l , l ' l ~ ' l ' l , ~ \ V ~1:'7.11..
~Ol~
TABLE
1. Species used in the molecular analysis with classilication. Classification of turhcllarians
folloiving Cannon (1986). GcxiBank accession numbers for the full 18s rRNA or rDNA sequences arc
given with an indication of which arc ticw sequences prcscntcd here (this study). Notes for duplicate
taxa: A Aguinaldo P t a/. ( 1 997); C-Carranza, Baguiii C(r Kiutort ( 1 997); K-Katayama, Nishioka Sr
Yamamoto (1 996)
A
c:
K
this studv
this studv
this study
this study
this study
c:
K
this stitdy
this study
this study
U4596 I
U70077
this study
this study
this study
this study
this study
this study
AJO I2520
this study
AJO12.514
this study
1185098
U70082
h i s study
AJOl2.508
this study
A J O 12507
this study
rontinuud
this stud)
KI
K2
this stud)
this stud)
this study
this study
AJ 2 28 7118
AJ228792
this study
this study
AJ228789
this study
.\J2223 7 9 6
rliir s ~ u d )
t h i s study
'\I2287 76
this study
.\J 228 7 7 3
this stud)
I)h4072
u270 I .?
Sequence alignment
Initially, we took aligned reference sequences from the SSU rRNA database at
the WWW rRNA server (URL http://rrna.uia.ac.be; Van de Peer et al., 1998) and
added sequences with ClustalW (Thompson, Higgins & Gibson, 1995) using default
nrl
1). ' 1 ~ .J.
. l , I ' l ~ ' l ' ~ . l ~ ~ \ fOi 7O, Il L
~.
weighting and gap penalties, and the profile alignment option. Bases that could
not be aligned unambiguously by eye and regions of the alignment involving
autapomorphic insertions greater than two bases were removed prior to phylogenetic
analysis. Wherever possible we selected regions of the alignment that began and
ended either with invariant bases or were identical in terms of purine (A/T) or
pyrimidine (G/C). Alignments were handled using GDE (Smith et al., 1994) for a
SUN workstation and exported to a Macintosh for phylogenetic analysis. Ambigously
aligned regions of our alignment were discarded prior to phylogenetic analysis. We
chose to omit three previously published full 18s rDNA sequences from our
alignment, and therefore analysis, as we detected sequencing errors and inconsistencies in regions of high sequence conservation; the taxa omitted were Bipalium
sp. (GenBank/EMBL accession D85086), Nematoplana sp. (D85093), and l4rticero.r
&mai (D85094), all fromKatayama, Nishioka & Yamamoto (1996).The full sequence
alignment used in these analyscs has been deposited with EMBL under accession
ds36895 and is available from The Librarian, The Linnean Society of London, or
via anonymous ftp from ftp.cbi.ac.uk under directory pub/databases/embl/align.
Phylogenetic anahhis
All analyses were conducted using PAUP" (PAUP 4.0.0d61 and 4.0.0d64;
Swofford, in press). For maximum parsimony analysis only consistency indices (CI)
excluding uninformative characters are presented. Wherever possible data scts were
bootstrap resampled (n= 1000). Molecular data sets were too large for branch-andbound and for these only heuristic searches were employed. Trees were rooted using
selected outgroups and character states were optimized using the ACCTRAN option,
i.e. reversals were preferred over parallelisms. Outgroups were chosen on the basis
of their alleged position as early branching bilaterians (according to the literature
e.g. A x , 1985, 1989; see also references in Littlewood et al., 1998).
Morphological data
Morphological data were analysed using the branch and bound option of PAUP"
(Swofford, in press). All 65 characters were equally weighed and unordered. Multistate taxa were treated as uncertain. The 26 ingroup taxa were rooted against five
outgroup taxa. Only the strict consensus trees were considered for interpretation
and branch support was determined using bootstrap resampling ( n = 1000; 'fast'
step-wise addition option due to time constraints with other search methods) and
in some instances the calculation of Bremer support indices (Bremer, 1994) using
AUTODECAY (v.2.9.9; Eriksson, 1997).
The compatibility of the most parsimonious solution, or the strict consensus of this,
with other phylogenetic solutions was tested using the non-parametric Templeton's
(Wilcoxon's signed rank) test (Larson, 1994).
Molecular data
We inferred phylogenies using two methods: maximum parsimony and minimumevolution distance method with the distance matrix calculated using a maximumlikelihood model (PAUP*, Swofford, in press; see also the paralinear/LogDet distance
method, Lake, 1991 ; Lockhart et al., 1994).
1N r E R R b L l I IONSHIPS O E 1’1A‘I”L’HCLI\lIN?’HES
89
With maximum parsimony we conducted heuristic searches (1 0 random addition
replicates) and weighted all characters equally. In all analyses gaps were treated as
a fifth character state. All trees involving the full 82 platyhelminth taxa were rooted
on an outgroup consisting of the Placozoa, Porifera, Ctenophora, Coelenterata (i.e.
the non-bilaterian taxa), Gnathostomulida, Nematoda, Gastrotricha, Nemertini,
Rotifera and Acanthocephala. This large array of outgroup taxa (1 9 sequences)
allowed rooting against diploblasts (8) and other triploblasts (1 1) thereby allowing
us to test for monophyly of the phylum.
For methods using distance matrices (NJ with minimum-evolution model) the
distance matrix was calculated using the maximum-likelihood option in PAUP*
(Swofford, in press). The transiti0n:transversion ratio, the gamma statistic and the
proportion of invariable sites were calculated using an initial NJ tree constructed
using a distance matrix calculated with the Log Det option. Each of these three
maximum-likelihood variables was calculated separately, its value then entered into
the model while the next parameter variable was calculated. This procedure was
repeated iteratively until the estiniates for each parameter variable (and the log
likelihood) did not change. A final maximum-likelihood model using these statistics
was then employed to estimate the molecular phylogeny.
Our optimal topologies, found by both distance methods and parsimony, were
compared to the morphology based phylogenies, presented herein, and previously
published morphological solutions (Ehlers, 1985a). Using MacClade (Maddison &
Maddison, 1992) we generated trees reflecting the alternative morphological hypotheses and tested the statistical significance of support afforded by the molecular
data on these alternative phylogenetic schemes using the Kishino-Hasegawa test
(Kishino & Hasegawa, 1989; implemented in PAUP”).
RESULI‘S AND DISCUSSION
Morphologv
Analysis of our morphological characters alone shows some similarities, but also
significant differences with Ehlers’s (1985a; Fig. 1) system. The strict consensus of
our 160 equally most parsimonious solutions is presented in Fugure 2a (length = 78,
CI = 0.800; RI = 0.9 19). Similarities are: (1) the basal position of the Catenulida and
Acoelomorpha; (2) the monophyly of the Acoelomorpha (Acoela plus Nemertodermatida); (3) the monophyly of the Neodermata; (4) the monophyly of the
Monogenea (Monopisthocotylea plus Polyopisthocotylea); (5) the monophyly of the
Aspidogastrea ( =Aspidobothrii) plus Digenea (collectivelyknown as the Trematoda);
(6) the monophyly of the cestodes (Gyrocotylidea, Amphilinidea, Eucestoda), and
(7) the monophyly of the Macrostomida and Haplopharyngida. The 500/0majorityrule consensus solution (Fig. 2b) further suggests the monophyly of the Cercomeromorphae (monogeneans plus cestodes) although with little support. Differences
are as follows: (1) the Neodermata are not the most derived group, but they are the
sister group of all the rhabditophoran turbellarians; (2) the Proseriata and Tricladida
do not constitute a monophylum; (3) the eucestodes are not the most derived group
within the cestodes; (4) Udonella and the other Neodermata form one clade (clade
comprised of Udonella and the Monogenea appears in the 50% majority-rule tree;
b. 50% majority rule consensus
a. strict consensus - 160 trees
GNATHOSTOMULIDA
NEMERTlNl
ROTIFERA
ACANTHOCEPHALA
GASTROTRICHA
NEMATODA
53
-
99
43
-
-
82
lchthyophaga
Udonella
Aspidogastrea
Digenea
Polyopisthocotylea
Monopisthocotylea
Eucestoda
Gyrocotylidea
Amphilinidea
Tricladida
Polycladida
Macrostomida
NEMERTlNl
25
0
GASTROTRICHA
-
53
1
44
-3.L
Udonella
Polyopisthocotylea
Monopisthocotylea
Eucestoda
Gyrocotylidea
Amphilinidea
lchthyophaga
Urastoma
0
0
2
12
0
0
Tricladida
43
1
bootstrap support
Bremer support
Lecithoepitheliata
Prolecithophora
Kalyptorhynchia
Ternnocephalida
Dalyelliida
Typhloplanida
Figure 2. (a) Strict consensus, and (b) 50% majority rule consrnsus tree of 160 equally most parGmonious solutions found with our morphological matrix; details
and matrix in Appendix 2. Trre length= 78, CI=0.808, RI=0.918. Bootstrap (percentage of 1000 replicates) and Bremcr support shown abo1.e and below
branches respectivrly.
Fig. 2b); (5) Fecampiida are not ‘dalyellioids’and the sister group of the Neodermata,
but close to the Proseriata; (6) Polycladida form ne clade with the Macrostomida
and Haplopharyngida (they are close to the last two taxa in Ehlers’ system but not
monophyletic with them); (7) Lecithoepitheliata, Prolecithophora, Kalyptorhynchia,
Temnocephalida, Dalyelliida and Typhloplanida form one clade, and (8) the last
four (50% majority rule; Fig. 2b) and three also form clades. Most importantly, the
ladder-like hierarchical structure of Ehlers’ system is replaced by a more ‘bush’-like
one.
We discuss supporting evidence for the various conclusions later in the context
of Figs 5 and 6 (combined DNA and morphological data).
Molecules
The full alignment of 101 18s rDNA and rRNA (published) sequences comprised
1358 unambiguously alignable positions, of which 995 were variable and 757
parsimony-informative.
Maximum parsimony found 168 equally most parsimonious trees (length = 5762;
CI = 0.338; RI = 0.6 17), of which the strict consensus is shown in Figure 3. Bootstrap
values were calculated with the ‘fast’ stepwise addition method (n = 1000). The
minimum-evolution distance tree was detcrmined using the following maximumlikelihood parameter estimates on the 1358 unambiguously aligned positions: proportion of sites assumed to be invariable = 0.1733, gamma distribution parameter =
0.7304, and transition/transversion (Ti/Tv) ratio = 1.63, ( - Ln likelihood = 26847.6).
Under this model the minimum-evolution score of the resolved tree (shown in Fig.
4) was 4.759. Bootstrap support, using the maximum-likelihood model as the distance
measure in a neighbour-joining search (n = 1000) is also shown on the solution.
Although the two methods of phylogeny reconstruction yielded different tree
topologies, there are some striking similarities between them. First of all, molecular
evidence suggcsts that the Platyhelminthes are a paraphyletic assemblage. The
separate appearance of the Acoela is the source of this irregularity although the
clade suffers from long-branching taxa. Both methods placed the acoels at or towards
the base of the Bilateria (Figs 3, 4). Other flatworms are more divergent, with the
gastrotrichs, gnathostomulids, rotifers and acanthocephalans separating them from
the acoels. Analysis of the data set with and without acoels had no effect on the
relative position of the other platyhelminth clades. However, the position of the
relatively long-branching acoel taxa may themselves have been influenced by
long-branching ingroup taxa through long-branch attraction (Felsenstein, 1978).
Establishing the position of the Acoela amongst the Metazoa, and relative to the
other platyhelminths, requires denser sampling of taxa to break up the long branches
(e.g. Aguinaldo et ul., 1997), but this is beyond the scope of the present study. The
relative position of the triploblastic outgroups is highly unstable but this is unimportant
as they act only to root the ingroup. With the distance method catenulids are sister
group to the Nemertini. Both methods resolve all other flatworms as monophyletic
with parsimony also uniting the catenulids. Of the taxa represented by two or more
sequences, most fell into recognizable monophyletic clades regardless of method
of phylogenetic reconstruction; namely the Acoela, Catenulida, Macrostomida,
Polycladida, Lecithoepitheliata, Kalyptorhynchia, Tricladida, Aspidogastrea, Digenea, Polyopisthocotylea, Monopisthocotylea (including Udonellu) and Eucestoda.
92
DIPLOBLASTS
NEMATODA
Awela
ACANTHOCEPHALA
p
c 92
q=Gnathastomula
Lineus
Prostome
LepidodermeNe
-Slenostomum
-A
ROTIFERA
GNATHOSTOMULIDA
NEMERTlNl
GASTROTRICHA
Catenulida
Macrostomida
Macrostomida
Polycladida
Prosenata
Prosenata
Nemertodermatida
Prosenata
Typhloplanlda
1
Kalyptorhynchia
Dalyellida
Temnccephalida
Typhloplanida
Prolecithophora
Fecarnpilda
Tnciadida
~
Aspidogestrea
Digenea
Polyopisthowtyiea
Monopislhowlylea
+Udonella
Gyrocotyildea
Eucesloda
rl-
k
Figure 3. Maximum parsimony rcsults with full 18s rDNA data set alone; strict consensus of 168
equally parsimonious trees showing full data set (left)and summary (right). Bootstrap support (percentage
of 1000 rcplicates) shown above branches ( 250%0 only); length = 5762; CI = 0.338; RI = 0.61 7. See
tcxt for further dctails.
At a higher level monophyletic Neodermata, Trematoda, and Cercomeromorpha
were also monophyletic. A number of important groupings were found to be nonmonophyletic, and of those densely sampled the Proseriata and Monogenea are of
particular interest.
T h c position of Jvemel-tinoides is highly problematic. Alone, it is rcsponsiblc €or the
apparent paraphyly of the Proseriata, and omitting this sequence from the analysis
keeps the proseriates monophyletic. Single sequences chosen as examplars of larger
clades are known to have profound effects on tree topology (Lanyon, 1985; Lecointre
et al., 1993). Traditionally, thc Nemertodcrmatida are considered the sister-taxon to
the Acoela (Ehlers, 1985a; Fig. 1) and clearly the group needs to be more densely
sampled.
The paraphyly of the Monogenea based on molecular data was first demonstrated
with partial 18s rDNA (Baverstock et al., 199 1) and most recently with partial 28s
rDNA (Mollarct rt al., 1997) but with a different relationship between the other
major ncodermatan groups. We discuss the monophyly of the monogeneans further,
below in connection with the morphological cvidencc.
Combined moqlzological and DNA evidence
Although the morphological and molecular data sets are suggesting two independent phylogenetic solutions, as indicated by the Kishino-Hascgawa test (Table
2), we have chosen to add the two data sets and determine the biological consequences
of any resultant phylogenetic solutions, in a true ‘total evidence’ approach (Kluge,
1989). Although there are convincing arguments for restricting a ‘total evidence’
approach to the addition of homogeneous indcpcndent data sets (Huelscnbeck, Bull
& Cunningham, 1996),it is only by adding the morphological data to our molecular
data set that we can map the biological consequences of a tree resolved by
incorporating all available evidence (see also the philosophy for ‘always combine’
in Huelscnbeck, Bull & Cunningham, 1996).
Maximum parsimony on the combined data (all characters unweighted, unordered)
yielded 6 equally most parsimonious trees (length = 5875, CI = 0.308; RI = 0.642);
strict consensus shown in Figure 5 with bootstrap values, and simplified in Figure
6 with unambiguous character changes mapped using MacClade; Maddison &
Maddison, 1992). The combined solution supports many of the conclusions based
on morphological evidence alone (Fig. 2). Thus, the Neodcrmata are monophyletic,
and within them the Monogenea (including Udonella which is monophyletic with the
Monopisthocotylea); the cestodes (Eucestoda plus Gyrocotylidea) and the trematodes
(Aspidogastrea plus Digenea) are monoplyletic as well. The sister group of the
Neodermata is a large ‘turbellarian’ taxon which, however, does not include the
polyclads, macrostomids or haplopharyngids, as it docs in Figure 2. Tricladida and
Proseriata are not monoplyletic, i.e. the taxon Seriata is invalid. As in Figure 2, the
Kalyptorhynchia, Typhloplanida, Dalyelliida and Temnocephalida constitute one
monophylum, with the kalyptorhynchs as the sister group of the others. The position
of the Nemertodermatida is unusual, appearing close to the Proseriata and not to
the Acoela, and the Acoela are not monophyletic with the ‘other’ Platyhelminthes.
As in Figure 2, Polycladida, Macrostomida and Haplopharyngida form one clade,
and the last two another one within it. The Catenulida are the most basal
platyhelminths.
The tree (Fig. 5) contains one serious inconsistency, i.e. the position of the
Nemertodermatida. Similarities of the complex system of the rootlets of epidermal
cilia and of the ciliary tips (Ehlers, 1985a) are strong evidence for a close relationship
of the Acoela and Nemertodermatida, although Xenoturbella, a genus unlikely to be
I ) . '".,J. l , l ' l ' ' l ' 1 , l ~ \ ~ 0 0 1l~i7.11..
$14
(a)
Trrchoplax
Mnamiwsrs
Beroe
ScVpha
Microcrwa
cm
i
51
-
Conwluta ulchra
Acbnvskra
1M
51
im
I
DIPLOBLASTS
Acoela
GASTROTRICHA
0GNATHOSTOMULIDA
0NEWTODA
Philodina
Mwilrfms
Neoschimhwchus
Centrorhmchus
67
8ROTIFERA
0
ACANTHOCEPHALA
ONENERTlNl
OCatenulida
0 Haplopharyngida
Macrostomida
0
0 Macrostomida
Polycladida
0 Proseriata
0 Nemertodermatida
0 Proseriata
Typhloplanida
Kalyptorhynchia
;
8
Dalyellida
0 Temnowphalida
0 Typhloplanida
0 UrastomaAchthyophaga
n
OFecampiida
Tricladida
U
Nematoplana
Lobstostma
Multicolyle
Schrstosma hamalobrum
Schrstosma spndala
SchrstosOma manswl
Froswhyncholdas
Teiracerasta
Hamnrmus
CalrcophorM
Oprsthorchis
G)fiauchan
Echirwstma
Fascrolopsis
Neopdysm
wplwnwdas
Fseudohexabofhrium
Kuhnia
Diclidophwa
Fsaudwnurraylrema
Leptm
calm$?
GVmdacWus
Wonella
GyrocoWe
Echrnmoccus
Abolhrium
Bothriocephalus
Grillofis
Roleoc halus
qrrmTa
liqurc 4. Scc
(qtioii
on fnt ing p q c .
OProseriata
OAspidogastrea
10
Digenea
U
Polyopisthocotylea
0
Monopisthocotylea
+Udonella
0 Gyrocotylidea
Eucestoda
Cmwluta pulchra
U
Acoela
GASTROTRICHA0
GNATHOSTOMJLIDA0
NEMaTODAO
8
ROTIFERA
PCANTHCCEPHALAO
0
NEMRTlNl
0
Catenulida
Haplopharyngida 0
0
Macrostornida
Macrostomida 0
U0
Polycladida
Lecithoepitheliata
Proseriata 0
Nemertodermatida 0
Proseriata 0
Typhloplanida 0
Kalyptorhynchia
0
8
Dalyellida
Ternnocephalida 0
Typhloplanida 0
Uraslornrulchlhyophaga 0
Fecarnpiida 0
Tricladida
I
Proseriata 0
Aspidogastrea 0
Digenea
Polyopisthocotylea
I
0
Manopisthocotylea
+Udone/la
Gyrocotylidea 0
n
Eucestoda
U
k-----i
0.01
Figure 4. Ncighhour-joininaiiiig tree using the minimum-c\~olutionHKY8.5 (maximum-likelihood) model
in PAUP* (Swolrord, in prcss), on full 18s rDNA data set; transition/trans\ersion ratio = 1.63; -In
likelihood = 26847.6: gamma ratc distribution with shape parameter = 0.73 1 ; proportion of invaria1)lc
positions = 0.173. Trec drziwn as (a)cladogram with bootstrap rcsampling pcrccntages ( n = 1000) shown
on 1)ranchcs; (I)) phylogram indicating long-l)ranch taxa, e.g. the arocls; scc text for furthcr details.
96
1). '1'. ,J. l , l ' l " l ' l , l ~ \ ~ O Of<T.If,.
l~
TABLE
2. Tree statistics ol'imcoiistraincd and constrainrd phylogenctic iolutions I'ound under parsimony
(heuristic searches). Constraints were takcn from morphological (this study and Ehlers, 1985) and
molecular (this study) solutions. Ixiigth of tree shown with consistency index excluding uninformativr
characters (CI),retention index (RI) and rcscalcd consistency index (RC:).'I rcprcsents the probability
applicablc; P<O.O5
that the constrained tree is cliflcrcnt from thr unconstrained solution; n/a-not
suggests that the constrained and unconstr;iincd solutions arc significantly diffcrcnt and argue fir
different phylogcnetic solutions; ***rrprcscnts RO.00 I . Iigurcs refer to topologies usrd to d
constraint
IZI
I'
IZC:
~.
71:
11/21
9i
***
I37
***
I li
***
~
11/a
***
***
***
a platyhclminth, and even other metazoans, show similarities with the Acoelomorpha
in thc epidermal cilia as well (Rohde, Watson & Cannon, 1988; Lundin, 1997, but
sec also Ehlers & Sopott-Ehlers, 1997a, N o r h &Jondelius, 1997), thereby somewhat
reducing the force of this argument (but see Lundin, 1997: bilayered dense plate
synapomorphic for the Acoela and Nemertodermatida). There is no morphological
evidence to suggest that the Nemertodermatida arc close to the Proseriata as shown
in Figure 4. Indeed, six morphological character losses (28, 31, 33, 50, 51, 53) and
four character gains (44, 45, 46, 47) would be required along the branch of
Nemertinoides in order to explain its position amongst the proseriates. We explain the
unusual position of the Acocla as the result of long-branch attraction (Felscnstein,
1978; Hendy & Penny, 1989), which leaves the closcly-related Nemertodermatida
in an abnormal position as well, and by the fact that only a single nemertodermatan
species was sequenced.
We discuss the synapomorphies of the various branches of Platyhelminthcs, and
recent literature on platyhelminth phylogeny, in the following.
Neodermata
Our findings on the monophyly of the Ncodermata confirm earlier findings by
Ehlers (1984, 1985a), Brooks, O'Grady & Glen (1985) and Rohde (1990) using
mainly morphological data, by Bavcrstock et al. (1991), Blair (1993) and Rohde et
al. (1993) using partial 18s rDNA sequenccs, and those by Rohde et al. (1995) using
, ,=
Tnchoplax
Mnemropss
Tnpedalia
Anemmia
Anfhopleura
scVpha
Miuonona
-
DIPLOBLASTS
Acoela
100
100
-
-
rStenoJtomum
A
StenostMlum -leumps
%nostomum - K
Stenostomum- C
Mamstomum
Mamsfwnum - C
Haprophawnx
Pammelostomurn
Mimstwnum
Micmstomum- C
mysanozwn
Pseudocems
Planm
Notoplena australis
Notoplanekoreane
Catenulida
Macmstornida
Haplopharyngida
Macrostomida
Polycladida
DlsmCBb
EKZgna
100
-
-
I bl
U
Planocsra - C
Nemstoplana
Nenmfiinoides
MonWrs
Archrlos
Otoplana
Paratopla
Dlssmrtlyndus
prangnella
a
-
Kalyptorhynchia
Typhloplanida
Fterastencola
Dalyellida
MrdalyeN,a
Temnocephels
Temnocephalida
Mesomstrada
Typhloplanida
BDthmmesostoma
Geocenfrophm sphyrocephsla
G-ntrophm
wwni
Lecithoepitheliata
Geocentrophm 6p
Kmnbrpra
Fecarnpiida
ldthyophaga
urnstoma
Urastorndchthyophaga
Ectoplana
Dendmmelopsrs
crenCh1a
Dendmmelum
Schmrdtea
Romankenkrus
Tricladida
Biosliurn
Aspidogastrea
Digenea
?-I
pl
Gyrocotylidea
Eucestoda
Monopisthocotylea
+ UdoneNa
Polyopisthowtylea
Figure 5.hlaximum parsimony results using full 18s rDNA data set arid morphological data ‘total
evidence’ solution; strict consensus of six equally most parsimonious trees sho\ving full data set (Icft)
only);
and summary (right). Bootstrap support @crcentagc of 1000 replicates) shown in boxes ( 250‘%1
length = 5875; C1 = 0.308; KI = 0.642).
1) I J 1J1 1 l . l , ~ ~ 0 0bar
1 ~I 1
DIPLOBLASTS
Amela
GNATHOSTOMULIDA
NEMATODA
GASTROTRICHA
NEMERTlNl
ROTIFERA
ACANTHOCEPHALA
Catenulida
Macrostomida
Haplopharyngida
Macrostomida
Polydadida
Pmseriata
Kalyptorhynchia
Typhloplanida
Dalyallida
Temnocephalida
Typhloplanida
I
1-
Ledthoepitheliata
In
Tridadida
4bij
3.10
Gyrowtylidea
characters changing unambiguously
Eucestoda
symbol from > to
n
0>1
-@-
1>0
Monopisthmtylea
+ Udone//a
Polyopisthocotylea
Figure 6. Summary of 'total evidence' strict consensus solution (see Fig. 5) with morphological characters
(numbered as Appc~tdix2) mappcd on branches. Only unambiguous changcs leading to the major
cladcs arc shown and arc indicated as character gains or losses-see figure for kcy. T h c position of
.?'emrrtinoidq representing the Ncmcrtodcrn~atida,is rcprcscntcd as a broken line lxcausc of its highly
anomalous position; scc tcxt for discussion and further details.
combined ultrastructural and DNA evidence. The monophyly of this taxon can now
he considered as beyond doubt. It is charactcrized by a number of convincing
synapomorphies (1, 2, 5, 7, 14, 20). However, our findings on the relationship of
the various neodermatan taxa with cach other differ from some of the earlicr studics.
According to Ehlers (1985a), as in our system, the Trematoda, comprised of the
Aspidogastrca and Digenea, is the sister group of all the other neodermatans, the
Monogenea is the sister group of the Gyrocotylidea, Amphilinidea and Ccstoidea
(Caryophyllidca plus Eucestoda), etc., but hi5 system is strictly hierarchical in the
scnse that in sequence, cach taxon is thc sister group of all the more terminal ones.
In contrast, in our system the cestodes and monogcneans are sistcr groups of ‘equal
rank‘. Ehlcrs docs not include Udonella in the Ncodermata. Brooks’ (1989) system
corresponds largcly to that of Ehlcrs (1 985a), but hc considers Udonella to bc the sister
group ofall thc other neodcrmatans. According to Blair (1 993), the Aspidogastrea may
be the moft basal taxon within the Neodermata, i.e. the sister group of all other
Neodermata. This suggestion is not supported by any othcr studics, including ours.
In our system, the neodcrmatans are comprised of two major cladcs, the trcmatodes
and thc Cercomeromorphae. A number of convincing synapomorphies for the
trematodes supports their monophyly, and one synapomorphy (prcsence of hooks,
unique in the Platyhelminthes and apparently secondarily lost in Udonella)supports the
monophyly of the Cercomeromorphae. Among the Ccrcomeromorphae, Lgyrocotylids
and cucestodcs form one clade, and the monogcneans the othcr. Importantly,
Udonella is shown to he a monopisthocotylean (which apparently has lost its hooks
secondarily) and the Monogenea are a monophyletic group. Inclusion of Udonella in
the Monogencans supports the carlier conclusion of Rohde (1996) and is explicit
evidence that the suggestion of Brooks, O’Grady & Glen (1985) and Brooks and
hlcLennan (1993a) of a sister group relationship of C‘donella with all the other
neodermatans cannot be accepted. Hence, their taxon Cercomeridca (allNeodermata
minus Udonella) is invalid. We will discuss the relative position of Udonella amongst
the Monopisthocotylca in a subsequent paper. Our finding furthcr supports the
conclusion of Rohde (1 996) that characters used for a phylogenetic analysis without
a prior liomoIo<gyanalysis (cf. Brooks & McLennan, 1993a) may yield wrong results,
especially in groups cxposed to different selection pressures, in this case a symbiotic
way of life.
Concerning the monophyly of the hfonogenea, our tree hascd merely on DNA
differs in this rcspect from the tree using combined morphological and DNA data.
In the DNA trccs, the Polyopisthocotylea are basal to the Monopisthocotylea, and
the latter to the cestodes, i.c. the two monogencan groups are closely related but
paraphyletic.
Sister group ofjGodermata
Ehlcrs (1984, 1985a) suggested that the sister group of thc neodermatans is to be
found among the ‘Dalyellioida’, and more specifically, that the ‘dalycllioid’ taxon
Fecampiidae may be this sister group. Evidence given for this suggestion was (1) a
free-swimming larval stage and (2) locomotory cilia with a single, cranial rootlet in
both groups. However, later studies have shown that a small vertical rootlet is indeed
present in at least one fecampiid species (Watson, Rohde & Williams, 1992; Watson,
Williams & Rohdc, 1992). On the other hand, ultrastructural studies by Watson,
Steiner & Rohde (1991), Watson, Williams & Rohde (1992), and Watson & Rohde
100
1) 1 ,J 1.1 I 11,I~b’OOl)I:7.11A
(1993a,b) have shown that there are similarities of spermiogenesis (proximodistal
direction of axonemes), protonephridia and eyes in both groups; studies using partial
18s rDNA did not show a close relationship of the fecampiid Kronborgia iJupodicula
with the neodermatans (Rohde et al., 1994). In our study, the fecampiid does not
appear to be close to the Neodermata; it is monophyletic with the parasitic
Ichthyophaga, which is usually included in the Prolecithophora (e.g. Cannon, 1986),
but unlikely to belong to it.
Brooks, O’Grady & Glen’s (1985) suggestion that Udonella is the sister group of
the other neodermatans, has been discussed above and must be rejected. Similarly,
their suggestion that the temnocephalids are the sister group of the Neodermata
(Udunella plus Cercomcridea) is without basis, as discussed in detail by Rohde
(1996). In particular, the ultrastructure of the protonephridia clearly shows that
temnocephalids are ‘dalyellioids’ (Rohdc, 1991). This conclusion was supported by
DNA studies of Rohde et al. (1993) and is supported by our findings (Fig. 5). The
taxon Cercomeria (Tcmnocephalida plus Neodermata) of Brooks, O’Grady & Glen
(1 985) must therefore be considered invalid.
Jondelius and Tholleson (1993), on the basis of a data matrix of largely superficial
characters whose homology is unlikely, suggested thatt a family of parasitic
‘dalyellioids’,the Pterasticolidae, is the sister group of the Neodermata. Rohde et al.
(1993) sequenced partial 18s rRNA of PteraJtericula and found that it is a ‘dalyellioid’
with no close relationship to the Neodermata. This finding is supported by our
study using complete 18s rDNA.
Rohde et al. (1993, 1995) suggested on the basis of partial 18s rDNA sequences
and protonephridial ultrastructure that a large taxon comprising most or all ‘turbellarian’ taxa is the much searched for sister group. This is suggested also by the
present study. All turbellarians except for the Acoela and Polycladida/Macrostomida/
Haplopharyngida and Catenulida are included in the sister group. In other words,
it includes all the Neophora in Ehlers’ sense: the large groups of parasitic Platyhelminthes have arisen very early in evolutionary history.
Sopott-Ehlers (1997) used ulstrastructure of female gametes to establish the
hypothesis that Prolecithophora and Rhabdocoela senm Ehlers (including the Neodermata) form a monophylum, the ‘Eulecithophora’. Although we have no DNA
sequences for the Prolecithophora, position of the ‘dalyellioids’ and the other
turbcllarians indicates that this hypothesis does not agree with our evidence (Figs
2-7).
Kornakova & JoRe (pers. comm.) have proposed the taxon Fecampiida for
the Fecampiidac and Notentera which have a ncodermatan-type of spermiogenesis
(axonemes of sperm directed from proximal to distal). They further argue: (1) that
all the Platyhelminthes with neodermatan-type spermiogenesis form a monophyletic
taxon, the Revertospermata, and (2) that the Fecampiida (spermiogenesis with
migration of the nucleus beside the incorporated axonemes within the sperm shaft,
no medial process) are the sister group to Urastomidae plus Neodermata (medial
process and proximo-distal fusion of axonemes within it). T o test this hypothesis,
we assumed that in the Fecampiida and Urastoma, for which we have DNA data,
proximo-distal fusion is indeed homologous with that of the Neodermata (i.e.
character ‘8’ becomes 1; character ‘7’ also becomes 1 assuming that an intercentriolar
body has been secondarily lost). The structure of the protonephridial flame bulb is
also assumed to be homologous in Urastoma, Fecampiida and Neodermata (i.e.
characters ‘14’ arid ‘15’ each become 1, and ‘39’ becomes 0), as is the acquisition
101
53
-
1
GNATHOSTOMULIDA
NEMERTINI
ROTIFERA
ACANTHOCEPHALA
GASTROTRICHA
NEMAT0DA
Figure 7. Strict consensus of 104 equally parsimonious solutions found with our morphological matrix
after recoding characters 7, 8, 14, 15, 20 for Fecampiida, Ichthyophaga and Urastoma (after Kornakova
& Joffe. pcrs. comm.; see text for full particulars). Tree length = 80, CI = 0.779, RI = 0.9 13. Bootstrap
(percentage of 1000 rcplicates) and Bremer support shown above and below branches respectively.
Asterisk indicates new cladc-compare with Fig. 2.
of a parasitic way of life (i.e. character '20' becomes 1). Analysis of the morphological
data matrix now shows that Fecampiidae, Ichthyophaga and Urastoma indeed form the
sister-group of the Neodermata as suggested by Kornakova & Joffe (our Fig. 7). The
tree now also shows a close relationship, i.e. monophyly of the Lecithoepitheliata,
Prolecithophora, Kalyptorhynchia, Temnocephalida, Dallyelida and Typhloplanida,
supporting evidence from the ulstrastructure of protonephridia. All these taxa have
a flame bulb with a weir formed by a single cell (Ehlers, 1989; Rohde, 1991,
further references therein) and except in the Lecithoepitheliata, ribs supported by
microtubules (Ehlers, 1989; Rohde, 1991, further references therein), although
several species of Prolecithophora have a different type of flame bulb (Ehlers &
Sopott-Ehlers, 199713; Watson & Rohde, 1997). In other aspects the tree does not
differ significantly from the previous analysis.
However, DNA data do not support this conclusion. Even in a tree, using
combined morphological and molecular data (six equally parsimonious trees; identical
topology as Figure 5, but length = 5879; CI = 0.307; RI = 0.641) Fecampiidae,
102
D. T. J. L I n m w o o D E T ~ I L .
Urastoma and Ichthyophaga do not appear as a sister-group of the Neodermata, and
the Lecithoepitheliata do not appear as monophyletic with the Prolecithophora,
Kalyptorhynchia, Temnocephalida, Dallyelida and Typhloplanida. This may indicate that similar spermiogenesis and ultrastructure of flame bulbs are not
homologous in the Fecampiidae/ Urastoma and Neodermata, and that similar ultrastructure of flame bulbs in Lecithoepitheliata is not homologous with that of the
Prolecithophora/Kalyptorhynchia/Temnocephalida/Dalyellida/Typhloplanida.
We know very little about the function of these two characters and parasitism in
these two groups may have led to convergence. Alternatively, differences in evolutionary rates may have affected the DNA component of the tree.
Seriata
Ehlers (1985a) kept the taxon Seriata of earlier authors, consisting of the Proseriata
and Tricladida, but pointed out that future studies would have to clarify whether it
is indeed monophyletic. Synapomorphies for the taxon given by Ehlers are a
characteristic pharynx of the plicatus type (pharynx tubiformis), and strongly follicular
gonads. As pointed out earlier, in the absence of comparative ultrastructural studies,
pharynx types are not useful for phylogenetic studies, and the second ‘synapomorphy’
is so general (and not exclusive to the Seriata) that a homology analysis is impossible.
Rohde et al. (1995), on the basis of partial 18s rDNA sequences and protonephridial
ultrastructure, concluded that the Seriata are not monophyletic and this was
supported by the study of Carranza, Bagufia & Riutort (1997). In our study, triclads
and proseriates belong to different clades, i.e. they are not monophyletic. It can
now be considered as very likely that Seriata is an invalid taxon, although inclusion
of the Maricola is recommended for future studies to vcrify this.
‘Dalyellioida’ and ‘Typhloplanoida’
Ehlers (1985a) considers both the ‘Typhloplanoida’ and the ‘Dalyellioida’ as
probably not monophyletic groups. Acording to him, a group within the ‘Typhloplanoida’ is likely to be the sister group of the Kalyptorhynchia. The ‘Dalyellioida’
include temnocephalids which have questionable monophyly, dalyelliids and fecampiids, and the Udonellida are provisionally included as well.
Watson, Steiner & Rohde (1991), Watson, Rohde & Williams (1992) and Watson
& Rohde (199313) had earlier shown on the basis of protonephridia, sperm and
spermiogenesis that the fecampiids do not belong to the dalyellioids, and this was
supported by the DNA study of Rohde et al. (1994). Our study supports this
conclusion. We show that kalyptorhynchs, typhloplanids, dalyelliids and temnocephalids represent one monophylum, and the last three taxa another. One convincing synapomorphy (38) supports monophyly of all 4 taxa, and two convincing
synapomorphies (35, 36) support monophyly of the last three. It is important to
note that the basal position of the Kalyptorhynchia with regard to the Dalyelliida/
Temnocephalida/Typhloplanida is supported by recent electron-microscopic studies
on protonephridial ultrastructure: in the kalyptorynchs, transitional stages between
the typical ‘rhabdocoel’ type of flame bulb and the type of flame bulb in other
turbellarians occur (Watson & Schockaert, 1998).
PoGycladida/Macrostomida/Haplophalyngida,Trepaxonemata
According to Ehlers (1 985a), the Rhabditophora (all Platyhelminthes excluding
the Acoelomorpha and Catenulida) consist of the Macrostomida and the rest, i.e.
INI’BKKEIhTIC>NSHII’S O F PIAATYHEI,I\1INI’HF,S
I03
the Trepaxonemata. Synapomorphies of the Trepaxonemata are (1) biciliated sperm,
(2) axonemes of sperm cilia of 9+‘1’ pattern (with complex central axis) and (3) ?
pharynx compositus. Rohde and Faubel (1 997) have shown that two centrioles
appear in early spermiogenesis of the macrostomid Parumalostomum fusculum, i.e.
possession of two cilia is not restricted to the Trepaxonemata, they have apparently
been secondarily lost in most macrostomids and the haplopharyngids. The second
character (lack of axonemes with 9 ‘ 1 ’ pattern) is purely negative, i.e. it may be
due to secondary reduction in macrostomids which lack fully developed cilia in
sperm. Such characters are of very limited use in phylogenetic analyses, as earlier
pointed out by Rohde (1 990, and above). The third character (pharynx compositus)
is also useless, because of the lack of ultrastructural studies, and hence lack of
evidence for the homology of various pharynx types (see above). In summary, there
is no evidence for the validity of the taxon Trepaxonemata. This is further supported
by our study which shows monophyly of taxa, some of which were included by
Ehlers in the Trepaxonemata, and others that were not.
Monophyly of the Macrostomida and Haplopharyngida, suggested by our study, is
supported by ultrastructural studies of Rohde and Watson (1998), who demonstrated
similarities in the terminal protonephridial complex of Haplophalynx rostratus and the
macrostomid Parumalostomum proceracauda, and by those of Rohde and Faubel (1997,
submitted), who demonstrated peculiar ‘hook’-like structures in the sperm of H.
rostratus and two species of Macrostomum. It is also supported by the finding of
Doe (1982) that a matrix syncytium of the copulatory stylet characterizes both a
macrostomid and haplopharyngid.
+
Catenulida/Acoela and Nemertodermatida
Ehlers (1985a) places the Catenulida as the most basal group of the Platyhelminthes,
and this is confirmed by this study. Synapomorphies supporting this placement are
(49, 50) for Catenulida plus the others; and (3) for the platyhelminthes excluding
the Catenulida. The last character, 9+‘1’ structure of the sperm axoneme, is
here to be assumed to have been secondarily lost in the Macrostomida and
Haplopharyngida (see discussion above: Trepaxonemata).
We consider the position of the Nemertodermatida and Acoela as unresolved in
our tree, in the case of the Acoela due to possible long-branch attraction, in the
case of the Nemertodermatida due to small sample size (single species). There is
convincing evidence (ultrastructure of tips of epidermal cilia and their rootlets, see
Tyler & Rieger 1977; but see Lundin, 1997: only bilayered dense plate possibly
synapomorphic for the Acoela and Nemetodermatina) that these taxa are closely
related. Our study suggests that the Platyhelminthes are not monophyletic, since
the Acoela are not included, but for the reasons just mentioned (the unresolved
position of Nemertodermatida and Acoela) this ‘suggestion’ must be considered
tentative and needs confirmation. Tyler & Tyler (1997), in a fascinating study,
have shown that various turbellarian taxa including the acoelans have epidermal
replacement and growth through immigration of deeper-lying cells. However, such
similarities are not necessarily due to monophyly, as pointed out by Rohde (1997).
Also, similar studies of other taxa (e.g. Xenuturbella, Gnathostomulida, Gastrotricha)
are necessary to rule out the possibility that this character is symplesiomorphic or
convergent. Ehlers & Sopott-Ehlers (1997a,b) have hypothesized that a septate
junction flanked by electron-light cisternae is a ‘ground pattern’ of the Platyhelminthes. We have decided against including this character in our data matrix
104
U I ,] I,ITTLEM'001) I T A L
because an intensive search for such structures in other invertebrates, to our
knowledge, has not been made and the possibility exists that it is either symplesiomorphic for many invertebrate phyla or a convergent character in various
small invertebrates.
In summary, our tree makes eminent sense. It supports many of the findings in
earlier studies. It supports the monophyly of the Neodermata, and of the Trematoda,
Monogenea and cestodes within them. It establishes a sister group relationship of
the Neodermata and a large group of turbellarians, the monophyly of a taxon
consisting of Kalyptorhynchida, Typhloplanida, Dalyelliida and Temnocephalida
and of a taxon consisting of the last three, it rejects monophyly of the Seriata but
establishes that Polycladida/Macrostomida/Haplopharyngida are monophyletic, as
are the last two taxa; as a consequence of this, validity of the taxon Trepaxonemata
is rejected. Our study agrees in some respects with the recent findings of Campos
et al. (1998), based on partial 18s rDNA sequences, who also found a sister group
relationship of Catenulida and the other platyhelminths (although the acoelans are
included in the latter) a non-'dalyellioid' position of the fecampiids, a sister group
relationship of the trematodes and the other neodermatans, and of the monogeneans
and cestodes.
Further studies must show the correct position of the Acoela and Nemertodermatids. It should also be stressed that morphological and molecular data are in
some respects contradictory, for instance concerning the position of the Fecampiidae/
Urastoma/Ichthyophaga and the relative position of the Lecithoepitheliata. Furthermore,
the incongruence between morphology and molecules implies conflict and necessitates
a reassessment or refinement of characters (Larson, 1994). Denser sampling of taxa
for molecular data, complementary sequences from independent genes, and inclusion
of additional morphological data are necessary.
ACKNOCL'1,EDGhfENTS
We are deeply indebted to the following individuals who provided us with material
for this study: Jaume Bagufia, Bjorn Berland, Rod Bray, Lori Colin, Anno Faubel,
Marco Curini-Galletti, Michelle Kelly-Borges, Delane Kritsky, Olga Raikova, Maria
Reuter, Marta Riutort, Oleg Timoshkin and Lynn Van Every. We are extremely
grateful to Lester Cannon for information on Urastoma and to Nikki Watson for
some critical comments on the data matrix, and to Rod Bray for comments on an
earlier draft of the manuscript. Two referees provided very useful and thorough
criticism, most of which we have incorporated into the final test. Elena Kornakova
and Boris Joffe (Zoological Institute RAS, St. Petersburg, Russia) very generously
shared their views on the morphology of Fecampiids, Urastoma and Ichthyophaga prior
to publication, and we thank them. David Swofford kindly provided a pre-release
version of PAUP* which was invaluable. Ian Ridgers provided expert technical
assistance running the automated sequencer. DTJL and KAC were funded by a
Wellcome Trust Senior Research Fellowship (0439652/95/2) to DTJL. K R was
funded by the Australian Research Council and the University of New England.
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~
Synapomorphics and autapomorphics (all ‘autapomorphics’ in Ehlcrs’ tcrminolo~gy)of the Platyhrlminthes taken from Ehlers (l985a). Only the important characters relevant to the current study
arc listed. For a complete list see Ehlers 1985a, pp. 169-176. (See Fig. 1).
Plathclminthomorpha: hermaphroditism, direct sperm transfer, internal fertilization and connrctcd
with it modified thread-like sperm.
I . Catenulida. Locomotory cilia distally with characteristic reduction of diameter of axonemc; protonephridium not paired; protoncphridial terminal cells with two cilia which have distinctly elongated
rootlets bending along the flame bulb; male gametes with characteristic bodies, but without cilia and
nucleus.
2. Euplathelminthes. 3-6 cilia/pm’ epidermal surface area; frontal glands.
3. Acoelomorpha. Characteristic complex pattern of rootlets of epidermal cilia; characteristic structure
of tips of epidermal cilia; lack of intestine lined by epithelium.
4. Ncmcrtodermatida. Statocyst with two statoliths.
5. Acocla. Characteristic pattern of rootlets of epidermal cilia; statocyst consists of 3 cells; hiciliated
sperm; during spcrmiogcnesis complete incorporation of axoncmcs in sperm bodies; characteristic
spiral-duet cleavagc.
6. Khabditophora. Lamellate rhabdites; duo-gland adhesi1.e organ; multiciliated terminal cells of
protonephridium.
7. Macrostomida. Characteristic duo-gland-adhesive organ; non-ciliated sperm. (The Haplopharyngida
arc considered to be part of the Macrostomida; their autapomorphy is a cranial probiscis-like
invagination of the tegument).
8. Trepaxoncmata. Biciliary sperm; 9 ‘ I ’ pattern of sperm axoncmc (complex central axis).
9. Polycladida. Characteristic plicatus-type pharynx; intestine with many lateral diverticula.
10. Ncoophora. O\,ary divided into germ and vitcllinc cells producing parts; cctolecithal egg.
1 I . Lecithocpithcliata. Complete reduction of‘ duo-gland adhesive organ; characteristic structure of
the ovary (germ cclls surrounded by vitcllinc cclls in the ovary).
12. Un-named taxon. Germovitellariuni; hlchlis’ gland; cilia of collar receptors surrounded by 8
microvilli.
13. Prolccithophora. Sperm with extensive membranous folds, nonciliated sperm; complete reduction
of duo-gland adhesive system.
14. Un-named taxon. Basal bodies of uniciliated collar r ptors intra- or subepidcrmal, with ringshaped root-like differentiation at level of basal bodies.
15. Seriata. Gonads strongly follicular; pharynx tubiformis.
16. Proseriata. Cranial rootlets of epidermal cilia converge terminating jointly at cranial margin of
epidermal cells; no lamellatc rhabditcs; weir (filtration apparatus of protonephridium) formed by two
cells.
17. Tricladida. Three-branched intestine; two germaria located at anterior end of gcrmo-vitclloducts;
formation of transitory embryonic pharynx.
18. Rhabdococla. Pharynx bulbosus.
+
‘Typhloplanoida’. According to Ehlcrs probably not monophylctic. Kalyptorhynchia monophyletic:
their autapomorphies cranial proboscis and complete incorporation of two axonerncs in sperm body
during spermiogenesis.
19. Doliopharyngiophora. Pharynx doliiformis at anterior end of body; complete reduction of duogland adhesive organ.
‘Dalyellioida’. Probably not monophylctic. According to Ehlers, monophyly of Tcmnoccphalida
questionable; Udoncllida are provisionally included in the ‘Dalycllioidea’, they probably do not belong
to the Ncodcrmata. T h r Fccampiidae belong to thc ‘Dalyellioida’ and may represent the sister group
of the Neodermata, because they have a frcc-swimming larval stage and the locomotory epidermal
cilia have a single, cranial rootlet.
20. Neodcrmata. Larval ciliated epidermis cast off and replaced by a syncytial neodermis with
subcpithclial pcrikarya; epidermal locomotory cilia with single, cranial rootlet; epithelial sensory
receptors with characteristic dense ‘collars’, weir of protoncphridium formed by two cells; during
112
spcrmiogcncsis complete incorporation of both axoncmcs in spcrm body; parasitic (or commcnsal) in
invcrtcl)rates.
2 1. Trcmatoda. Ciliatcd cpidcrmal ccls of larva scparatcd tiy cytoplasm of thc ncodcrmis; male
copulatory organ is a cirrus; mollusc invcrtchratc hosts.
22. Aspidobothrii. 1,an.a (cotylocidium) with vcntro-caudal sucker which becomcs a Iargc alvcolatcd
adhcsivc organ in the adult; fcw ciliatcd cclls of 1an.a; ncodcrmis with charactcristic microvilli ( =
microtubcrcles); oviduct scptatc (,\lam.@, MulticoQk, StichocoQlt, Rugogactu prol)ably do not belong to
the aspidobothriins).
23. Digcnca. Vegctativc multiplication in mollusc host; ciliatcd cpidcrmal cclls of miracidium arranged
in regular traiisvcrsc rows; inclusion of gnathostomous vcrtchratc in lifc cyclc; ccrcaria.
24. Cercomcromorphac. Postcrior hook of larva and adults (primarily probably 16 hooks).
25. hlonogcnca. Ciliatcd cpidcrmal cclls of l a n a (primarily 60 cclls) arrangcd primarily in 3 complcxcs
(anterior, middle and caudal); two pairs of pigmcntcd rhabdomcric photorcccptors; inclusion of
gnathostomous vcrtcbratc in lifc cyclc (pcrhaps already carlicr; in 24); loss of invertebrate host.
26. Ccstoda. Ciliatcd cpidcrmis of larva syncytial; 10 caudal hooks; loss of cntodcrmal digcstivc system;
rcticular protoncphridial systcm, male copulatory organ a cirrus; inclusion of vcrtchratc in life cyclc
(two host cyclc, pcrhaps already in 24); cndoparasitic in vcrtclxitc.
27. Gyrocotylidca. Caudal roscttc organ; apical proboscis.
28. Ncphroposticophora. Caudal opcning of protoncphridium.
29. Amphilinidca. Extreme Icaf-likc shape; charactcristic apical organ.
30. Ccstoidca. Six caudal hooks; larva (oncosphcrc) without ncnjous systcm and cpithclial sensory
rcceptors; microtrichcs of ncodcrmis; spcrm without mitochondria.
3 1. Caryophyllidca. Uniciliatcd spcrm; during spcrmiogcncsis 110 incorporation of axoncmc in spcrm
body; invertebrate host is an annclid.
32. Euccstoda. Syncytial cpidcrmis of oncosphcrc larva with spccial protcin inclusions; loss of caudal
hooks at mctaccstoid stagc; scvcral sets of gonads and gcnital structurc; hcad primarily \vith tiothria.
APPENDIX 2
Morphological character definitions uscd to reconstruct phylogenctic relationships Iictwccn the
major platyhclminth taxa. Scc main text for further cxplanation and sources. Prcscncc of character
described codcd as I ; abscncc codcd as 0. Taxa where homoloLgywas not confirmed for a particular
charactcr wcrc codcd as O / 1 and were treated as uncertain in the analyses.
O/ 1 to be run as '0' or ' 1' Iiccausc thcrc is a strong possibility that characters arc not homologous; ? =
missing data. Notc: pharynx structurcs have not Ixcn uscd because of insufficient evidencc for homoloq
of various types in different taxa.
Nodennata onb (except possibly 7 and 14); some similar characters in othcr Platyhclminthcs and
lower iiivcrtcbratcs arc probahly not homologous.
1. Cilia or larval cpidcrmis with single horizontal (cranial or rostral) rootlet.
2. Larval epidermis at cnd of free-living larval stagc replaced b y syncytial tccgumcnt (ncodcrmis)
conncctcd to subsurface perikarya by branching processes.
3. Epidcrmal ciliatcd cells of larva with intracpithclial nuclci and separated from each othcr by
ncodcrmis.
4. Syiicytial ciliatcd cpidcrmis of lama, with intracpithclial nuclei.
5. Scnsory rcceptors with characteristic (neodermatan type) clcctron-dcnsc collars.
6. Two pairs (somctimcs one pair) of pi,pentcd occlli in oncomiracidium.
7. During spcrmiogciicsis, incorporation of axonemcs into spcrm body by proximo-distal fusion,
intcrcentriolar body.
8. Modificd proximo-distal incorporation of spcrm axoncmcs (lack of interccntriolar body) ( M o l l ogcnca, IJdonellu and Kiunbogia only) (in K'ronbov'u possibly not homologous with thc Ncodcrmata,
hence O / I).
9. Crested-like body (bodies) in spcrm (in the onc monopisthocotylcan found possibly not homologous
with the ccstodcs, hence O/ 1).
10. Malc copulatory organ a cirrus (only in the Trcmatoda, prol)ably not homologous in othcr
neodermatans).
1 1. Copulatory organ a pciiis or penis stylct.
12. Large leaf-like body, lack of proglottids (Ccstoda onlyj.
13. \'itelloducts lined t)y discrete cells.
14. Flame h i l l ) formed by t\vo cells, a terminal and proximal canal cell, \vcir of flame l ~ u l hconsists
of two rows of longitudinal ribs. i.e.. outgrowths of the terminal and the proximal canal cells (in
non-ncodcrinat~~ims
possibly not homologous, hence O/ I ) .
15. Ncodcrmatan type flame bull) ( 14j, and ~~rot"ilepliridialcapillary uith scptatc junction.
16. Paired posterior excretory pores in lana.
17. Posterior excretory pore in adult.
18. Paired anterior excretory pores in l a n a and adult.
19. Ncodcrmatan t)rpr of Hamc hull) (1 4), and protoncphridial capillary u3hout scptatc junction (in
the one monopistliocolyleaii found possibly not homologous, hcncc O/ I).
20. Always parasites of in\wtcl)ratcs and/or vertcl)ratcs.
2 1. Vcrtclxitc host only ([,?/ondo assumed to I)c primarily a \.crtc.l)ratc parasite).
22. Invertebrate (mollusc) and facultative or ol)lig;itc \w-tehratc host.
23. Posterior or \.cntral sucker delimited from parcnchyma by distinct capsule.
24. il'cll defined posterior attachment organ (haptor), liut not separated from parenchyma by capsulc.
25. L a n a (and sometimes adult) with hooks (perhaps lost secondarily in tidonella, hcncc O/ I).
26. 'I~cnhooks.
27. No intestine (Ccstoda only, prol)al)ly convcrgcntly evolved in Fccampiida, hence O / 1j.
I'laphtlrriintiit.\ onh
28. Fcmalc gcrmarium divided into ovary and vitcllarium, or common gcrmarium di\.ided into egg
and yolk cell produciiig parts, rctolccithal egg.
29. Fcmalc gcrniarium not divided into ovary and vitcllariurn, or common gcrmarium not divided
into egg arid yolk cell producing parts, cndolcc.ithal egg.
30. Lamcllatcd rhahditcs.
3 1. Duo-gland adhesive organ.
32. Macrostomid-Imaplopharyiigid typc of duo-gland adhesive system.
33. 9 ' 1' structure of sperm axonelncs (perhaps secondarily lost in the hlacrostomida and Haplopharyngida, hence O / 1).
34. Spcrm with 'bristles'.
Characteristic type of spcrmiogcncsis and spernm (dense heel, rotation of flagella, spur in mature
sperm).
36. Spcrm with ro\v of characteristic (tcmnoccphalid, dalycllid, typhloplanid) dense granules.
37. Single-cell flame bulb (in the Catcnulida prol)al)ly indcpendcntly cvol\wl in view of the many
other differences, hence O / 1j.
38. Ribs of single cell Hamc bulb supported by microtuhulcs.
39. Two-cell flame bulb, weir formed by interdigitations of outgrowths of terminal and proximal canal
cells (possibly homologous with 14).
40. Weir of flame bulb formed by interdigitations of outgroivths of the terminal cell.
41. Single cell Hamc liulh with single row of longitudinal ribs.
42. Horizontal rootlcts of epidermal cilia con\wgc, terminating at the cranial margin of the epidermal
cclls (possibly not homologous in the Proscriata and hlacrostomida, hence O/ 1).
43. Anterior end with prominent rhabdoid tracts or a protmscis (tracts and proboscis possibly not
homologous, hcncc O/ 1j.
44. Distal part of cpidernmal cilia with characteristic 'acoelomorph-type' reduction in number of
axoncmal microtubules.
45. Rootlets of epidermal cilia with characteristic 'acoclomorph-type' complex pattern, and bilayered
dense plate.
46. Pulsatile bodies.
47. Acoclomorpha type of frontal organ.
48. Matrix syncytiuin of copulatory stylct.
49. Neoblasts (stem cells) that give rise to all differentiated cell types (differentiated cells do not
proliferate).
50. Kostral-caudal/vertical rootlet system of cpidcrmal cells without accessory ccntriolcs.
+
51. Dcnsc cxtraccllu1;ir matrix as true hasal lamina of ccto-and cndodcrm.
52. Spiral clca\ugc with two or four quartcts of micromcrcs and mcsodcrm out of micromcrc 2d or
-M or probahly derived from this paitcrn.
53. Intcstinc lined by cpithclium (or probably sccondarily lost).
54. True hindgut with anal opcning (it is douhtlul that the Gnathostomulida have a true anus, hcncc
o/ I).
55,
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
Wcll dcvclopcd brain and orthogon (Iiossilily sccondarily lost in the Acoclomorpha, hcncc O/ I).
Protoncphridia prcscnt (possihly secondarily lost in the Acoclomorpha, hcncc O/ I).
Charactcristic sclcrotizcd chewing apparatus of Gnathostumulida and Rotifcra.
hlonociliatcd cpidcrmis.
Spcilic bilatmtl clca\agc of C;astrotrirha and Ncmatda.
Charactcristic cpicuticlc mcnihranc or single hilaycr of cuticle of Gastrotricha and Ncmatodii.
Radially symmctric pharynx with niytrpithclial cclls (inrwtcd Y-lumcn).
Basic*pitliclialcirriimrntric brain of (;astroiricha and Ncmatoda.
Ncuropileous ncn'c ring in a sihtcrminal position.
hlostly intraccllular cuticle.
Charactcristic crn1)ryolo.g;): with movcmcnt of apical clcavagc pole from posterior to anterior end.
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