REVIEW / SYNTHÈSE

Molecular phylogeny of the Platyhelminthes1

Jaume Baguñà and Marta Riutort

Abstract: The phylum Platyhelminthes has traditionally been considered the most basal bilaterian taxon. The main dif-ficulty with this placement is the lack of convincing synapomorphies for all Platyhelminthes, which suggest that theyare polyphyletic. Recent molecular findings based on 18S rDNA sequence data and number and type of Hox genesstrongly suggest that the majority of Platyhelminthes are members of the lophotrochozoan protostomes, whereas theAcoelomorpha (Acoela + Nemertodermatida) fall outside of the Platyhelminthes as the most basal bilaterian taxon.Here we review phylum-wide analyses based on complete ribosomal and other nuclear genes addressed to answer themain issues facing systematics and phylogeny of Platyhelminthes. We present and discuss (i) new corroborative evi-dence for the polyphyly of the Platyhelminthes and the basal position of Acoelomorpha; (ii) a new consensus internaltree of the phylum; (iii) the nature of the sister group to the Neodermata and the hypotheses on the origin of parasit-ism; and (iv) the internal phylogeny of some rhabditophoran orders. Some methodological caveats are also introduced.The need to erect a new phylum, the Acoelomorpha, separate from the Platyhelminthes (now Catenulida +Rhabditophora) and based on present and new morphological and molecular characters is highlighted, and a proposalmade.

Résumé : Le phylum des plathelminthes est traditionellement considéré comme le taxon le plus basal des organismesbilatéraux. Le problème principal avec ce positionnement est l’absence de synapomorphies convaincantes pour tous lesplathelminthes, ce qui laisse croire que le groupe est polyphylétique. Des observations moléculaires récentes basées surdes séquençages de l’ADNr 18S et sur le nombre et les types de gènes Hox fournissent de forts indices que la plupartdes plathelminthes font partie des protostomiens lophotrochozoaires, tandis que les acoelomorphes (acoeles + nemerto-dermatides) sont exclus des plathelminthes et forment le taxon le plus basal des organismes bilatéraux. Nous faisons lasynthèse des analyses faites à l’échelle du phylum et basées sur l’étude des gènes complets des ribosomes et d’autresgènes nucléaires afin de répondre aux principales questions qui concernent la systématique et la phylogénie des plathel-minthes. On trouvera donc ici une présentation et une discussion de (i) nouveaux indices qui corroborent la polyphyliedes plathelminthes et la position basale des acoelomorphes; (ii) un nouvel arbre consensus interne du phylum;(iii) l’identité du groupe soeur des néodermates et les hypothèses sur l’origine du parasitisme et (iv) la phylogénie in-terne de quelques ordres de rhabditophores. Un certain nombre de mises en garde méthodologiques sont aussi formu-lées. Nous mettons en évidence la nécessité de créer un nouveau phylum, les acoelomorphes, distinct desplathelminthes, qui comprend maintenant les catenulides et les rhabditophores, d’après des caractéristiques morphologi-ques et moléculaires, actuelles et nouvelles. Nous en faisons la proposition.

[Traduit par la Rédaction] Baguñà and Riutort 193

Before cladism: An alternative phylogenetichypothesis for the phylum Platyhelminthes

Since its erection by Gegenbaur (1859), the phylumPlatyhelminthes has played a leading role in discussions ofmetazoan phylogeny, its simple body form and lack of de-rived features turning them into an “ideal” intermediateclade between radial cnidarians and the more complex and

advanced bilaterians. In the precladistic era, presence/ab-sence of a true coelom was the main issue of discussion re-garding the origin and evolution of bilaterian metazoans.Accordingly, whether the lack of a true coelom (e.g.,acoelomates or pseudocoelomates) was considered primitiveor secondarily derived was the key to position the acoelo-mate Platyhelminthes. The majority of zoological textbooksadopted the view, after the influential work of Hyman

Can. J. Zool. 82: 168–193 (2004) doi: 10.1139/Z03-214 © 2004 NRC Canada

168

Received 26 March 2003. Accepted 6 August 2003. Published on the NRC Research Press Web site at http://cjz.nrc.ca on 16 April2004.

J. Baguñà2 and M. Riutort. Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028Barcelona, Spain.

1This review is one of a series dealing with aspects of the biology of the phylum Platyhelminthes. This series is one of severalvirtual symposia on the biology of neglected groups that will be published in the Journal from time to time.

2Corresponding author (e-mail: bagunya@bio.ub.es).

(1951), that the lack of a coelom was the primitive bilateriancondition. Therefore, Platyhelminthes were considered anearly-emerging clade forming the likely sister group of allthe other Bilateria, which themselves were divided into thetwo coelomate superclades protostomes and deuterostomes.Moreover, the pseudocoelomates, which comprised an ill-defined polyphyletic assemblage of phyla and referredsometimes as the “Aschelminthes”, were often considered anintermediate phylogenetic stage between acoelomates andtrue coelomates (Fig. 1A). The view of acoelomates asearly-emerging bilaterian clades was linked to the followingspecific hypothesis/theory as to how bilaterians originatedfrom radially constructed organisms (e.g., cnidarians andctenophores): the planuloid–acoeloid hypothesis (von Graff1882). According to the hypothesis, the first bilaterian, simi-lar to present-day acoel platyhelminths, originated from a ra-dial creature similar to the extant planula larva of cnidarians.

A rather different view was based on the idea that thegastral pouches of cnidarians are homologous with thegastral pouches (enterocoels) which form the coelomic cavi-ties of deuterostomes (see Willmer 1990; for general refer-ences see Rieger and Ladurner 2001). Therefore, features ofdeuterostome development were assumed to be primitiveamong Bilateria. Under this scheme, protostome develop-ment features (e.g., spiral cleavage) were derived, coelomwas of early origin, and Platyhelminthes originated fromcoelomate ancestors by reduction of coelomic cavities in theadult (Remane et al. 1980) or by progenesis from larvalforms (Rieger 1985). Lack of a one-way digestive systemand of circulatory and respiratory systems in Platyhelmin-thes were also considered features derived by secondary lossfrom more complex coelomate ancestors (Fig. 1B). Such a

view also implied a completely different view on bilaterianorigins. This was encapsulated in the Archicoelomate The-ory, which derives from Haeckel’s gastrea (1874) and itsmodern versions: the bilaterogastraea (Jägersten 1955) andthe cyclomerism (Remane 1963) theories. Both posit a firstbilaterian with coelomic cavities that originated from gas-tric pouches of pelagic/benthic, larval/adult gastraea-type,cnidarian-like organisms. To circumvent some problemsposed by the last two theories, some authors (Nielsen andNorrenvang 1985; and Rieger 1994) postulated a bilaterianancestor with a biphasic life cycle, with a microscopicacoelomate/pseudocoelomate pelagic larva that by progen-esis gave the main acoelomate/pseudocoelomate clades anda benthic macroscopic coelomate adult that radiated into themain coelomate groups. However, how such a complex, in-direct developing, bilaterian ancestor evolved from a rathersimple radial organism with direct development has neverbeen satisfactorily explained.

The advent of cladism: Which is the sistergroup to the Platyhelminthes?

The advent of cladism promised to introduce a more ratio-nal basis regarding the taxonomy and phylogeny of Platy-helminthes. Cladism uses large sets of derived inclusivecharacters or synapomorphies under strict rules of phylogen-etic reconstruction. Accordingly, throughout the last third ofthe past century many different phylogenetic placementsbased on adult and embryo morphological characters wereproposed for the Platyhelminthes (for a short summary seeTyler 2001). Prominent among them were the following:(i) sister group to the Gnathostomulida forming the Platel-

© 2004 NRC Canada

Baguñà and Riutort 169

Fig. 1. Conflicting traditional phylogenies of the Metazoa and on the origin of Platyhelminthes and the acoelomate condition. (A) Anevolutionary tree based on the principle of gradual increase in complexity and assuming the acoelomate condition as primitive withinthe Bilateria, with pseudocoelomates forming a transitional state towards the coelomate groups Protostomia and Deuterostomia (afterHyman 1951). (B) An evolutionary tree based on the proposal that the acoelomate and pseudocoelomate conditions arose by secondaryreduction of the coelomic cavities (pseudocoelomates) or through progenesis (Platyhelminthes) from protostomian coelomatic ancestors(after Remane et al. 1980; Rieger 1985). In both schemes, coelomic cavities had a monophyletic (C) or a diphyletic (C′ ) origin.× marks the loss of coelomic cavities in B.

mintomorpha (Ax 1985, 1996; Eernisse et al. 1992; Zrzavyet al. 1998); (ii) sister group to the Gnathifera (Ahlrichs1995); (iii) sister group to the Nemertea forming the Par-enchymia (Nielsen 2001); (iv) sister group to a clade of dif-ferent spiralian groups (Brusca and Brusca 1990; Zrzavy2001); and (v) as a paraphyletic clade at the base of theBilateria (Haszprunar 1996a, 1996b). With the exception ofthe last one, all consider Platyhelminthes a monophyleticclade placed within, not at the base of, the protostomianbilaterians. However, the majority of characters used (e.g.,hermaphroditism, filiform sperm, internal fertilization, bi-flagellate sperm, loss of acrosome, and loss of prototrochand metatroch) likely represents symplesiomorphies or homo-plasies. Other characters, such as typical spiral cleavage andladder-like nervous system (Brusca and Brusca 1990), wereused but scored a too limited range of taxa. Problems plagu-ing cladistic analyses (e.g., character selection, characterconstruction, reconstructing ground-patterns, character weight-ing, and taxon selection and level of analysis) have beenthoroughly discussed and aptly summarized in Jenner andSchram (1999).

Among the characters advanced as potential autapomor-phies for Platyhelminthes, only two of them (i.e., lack of mi-tosis in somatic cells and lack of anus) bear some potential.Absence of mitosis in epidermal and other somatic cells(Ehlers 1986; Ax 1996) refers to the inability of these cellsto divide, worn out cells being replaced by undifferentiatedcells that are known as neoblasts in Platyhelminthes (Baguñà1981; Baguñà et al. 1989). However, this character is equiv-ocal and very poorly stated because most somatic cells aredifferentiated and, unless they dedifferentiate, never divide.Moreover, it has not been properly scored in many phyla.Finally, lack of anus interpreted as a secondary loss remainsthe only potential unique synapomorphy if Platyhelminthesare nested deeply within the Bilateria, which does not seemto be the case (see below). Other phyla such as Gnathost-homulida and Gastrotricha need to be reassessed withregards to this character. Homology between the anus andhindgut in Gnathosthomulida and Micrognathozoa to thoseof other bilaterians is still contentious (Knauss 1979;Haszprunar 1996b; Littlewood et al. 1999a; Kristensen andFunch 2000), and the distribution of hindguts within Gastro-tricha suggest an evolution within the phylum (Hochbergand Litvaitis 2001).

The unsettled question: Is thePlatyhelminthes a monophyletic group?

Proving the monophyly of Platyhelminthes is tantamountto finding their likely sister group. Traditionally, the Platy-helminthes have been considered a single phylum (Hyman1951) and within them, three clearly monophyletic groupsare recognized (Karling 1974; Ax 1985, 1996; Ehlers 1985;Smith et al. 1986) (Table 1): the Acoelomorpha (Acoela +Nemertodermatida, Ehlers 1985), the Catenulida, and theRhabditophora (Ehlers 1985), which comprises all otherPlatyhelminthes including the parasitic classes (Fig. 2A,2B). Autapomorphies of the Acoelomorpha are a specificstructure of its basal body-rootlet system of cilia, bent cili-ary tips, and fine structure of frontal organs (Smith et al.1986). Another suggested autapomorphy, absence of proto-

nephridia (Ehlers 1985; Ax 1996), has to be assessed cau-tiously because being a negative character could also be in-terpreted as an extant plesiomorphy, whereas the specialcleavage type of acoels, the duet-type, has not been unequiv-ocally coded in nemertodermatids. Catenulida is character-ized by an unpaired, dorsomedially located protonephridium,testes and male genital pore located dorsally in the anteriorhalf of the body, and aciliary, nonmobile sperm. Presence oflamellate rhabdites (solid secretions, or rhabdoids, with acortical layer composed of concentrically arranged lamellaand a medulla that varies from homogeneous to granular; Ax1996), duo-gland adhesive organs, and paired protonephridiawith flame bulb and canal cell interdigitating to form a weir(Ehlers 1985) are the main autapomorphies of the Rhab-ditophora.

Although the synapomorphies support each one of thesethree clades, the lack of robust morphological synapomor-phies uniting these three major clades was the main argu-ment advanced by Smith et al. (1986) to question themonophyly of this group. However, although they could notdemonstrate the monophyly of the phylum, which they be-lieved to be monophyletic, they left the three main cladesunconnected until such time as new synapomorphies were

© 2004 NRC Canada

170 Can. J. Zool. Vol. 82, 2004

Phylum Platyhelminthes Minot, 1876 (Hyman, 1951) (synonyms:Platyelmia Vogt, 1851; Platyelminthes Gegenbaur, 1859;Plathelminthes Schneider, 1873 (Ehlers, 1985))Acoelomorpha Ehlers, 1985

Order Acoela Uljanin, 1870Nemertodermatida Karling, 1940

Catenulida Graff, 1905Rhabditophora Ehlers, 1985

Order Macrostomida Karling, 1974Haplopharyngida Karling, 1974Polycladida Lang, 1881“Lecithoepitheliata”* Reisinger, 1924Prolecithophora Karling, 1940Rhabdocoela Meixner, 1925

Suborder “Dalyeillioida”* Bresslau, 1933Kalyptorhynchia Graff, 1905“Typhloplanoida”* Bresslau, 1933Temnocephalida Blanchard, 1849Revertospermata Kornakova and Joffe, 1999

Genostomatidae (genus Ichthyophaga)†

Notonteria (genus Notonteria)†

Urastomidae (genus Urastoma)†

Fecampiida (genus Kronborgia)†

Neodermata‡ Ehlers, 1985Order Seriata Bresslau, 1933

Suborder Proseriata Meixner, 1938Bothrioplanida Sopott-Ehlers, 1985Tricladida Lang, 1884

Note: Current classification is based on Karling (1974), Ehlers (1985),Smith et al. (1986), and Kornakova and Joffe (1999).

*Clades that are likely not monophyletic.†These four clades cluster together in a group yet to to be given a for-

mal name, although it is informally called the INUK (Ichthyophaga–Notonteria–Urastoma–Kronborgia) clade (Littlewood et al. 1999a, 1999b).

‡Includes the traditional parasitic classes Trematoda (Aspidogastrea +Digenea) and Monogenea and Cestoda (which nowadays, since Ehlers1985, belong to the Rhabditophora).

Table 1. Current classification of the Platyhelminthes.

© 2004 NRC Canada

Baguñà and Riutort 171

Fig. 2. Evolutionary trees depicting the relationships among the main free-living and parasitic orders of Platyhelminthes based on mor-phological characters according to Karling (1974) (A), after Ehlers (1985) (B), and according to Smith et al. (1986) (C). Three mainclades are well defined: Acoelomorpha, Catenulida, and Rhabditophora (which also includes the parasitic class Neodermata). Note thatthe Platyhelminthes are monophyletic in Figs. 2A and 2B, whereas they are polyphyletic in Fig. 2C. (D) Relationships of a paraphyle-tic Platyhelminthes within the Metazoa (after Haszprunar 1996a). Main apomorphic characters defining the three main groups aremapped onto the trees. The characters used to generate the trees in Figs. 2A, 2B, and 2C are as follows: 1, specific structures of itsbasal body-rootlet system; 2, bent ciliary tips; 3, fine structure of frontal organs; 4, unpaired dorsomedially located protonephridium; 5,testes and male genital pore located dorsally; 6, aciliary sperm; 7, presence of lamellate rhabdites; 8, duo-gland adhesive organs; and9, paired protonephridia with flame bulbs. The characters used to generate the tree in Fig. 2D are as follows: a, specific structures ofits basal body-rootlet system; b, bent ciliary tips; c, pulsatile bodies; d, four or more cilia in terminal cells of protonephridia; e,lamellate rhabdites; f, nerve ring around ventral part of pharynx; g, unpaired dorsomedially located protonephridium; h, testes and malegenital pore located dorsally; i, aciliary sperm; j, preformed anus; k, loss of frontal glandular complex; and l, testis sacular. Dottedlines indicate uncertain phylogenetic placements. See text for more details.

found that supported their believes (Fig. 2C). Moreover, theydid not provide an alternative hypothesis for the sister groupof the three clades. In striking contrast, Haszprunar (1996a,1996b) found morphological support for the paraphyly ofPlatyhelminthes, with acoelomorphs branching out first, fol-lowed by rhabditophorans and catenulids (Fig. 2D). Interest-ingly, acoelomorphs were considered by Haszprunar (1996a,1996b) to be stem-group bilaterians, forming the sister groupto the remaining extant bilaterian clades. In Haszprunar’sproposal, the distinctive characters supporting the mono-phyly of the sister clade of the Acoelomorpha were a centralnervous system with paired cerebral ganglion at the anteriorend and a protonephridium (Haszprunar 1996a); other char-acters were further added (Haszprunar 1996b): orthogonalnervous system, spiral quartet cleavage, and fixed cell fateduring cleavage. Closer inspection and further testing ofthese characters in ulterior cladistic analyses (Zrzavy et al.1998; Littlewood et al. 1999a; Peterson and Eernisse 2001)did not support the paraphyly of Platyhelminthes and thebasal position of Acoelomorpha. Haszprunar’s proposal hadmerits because it reconsidered some acoelomorph characters(e.g., lack of protonephridia and lack of cerebral ganglia andorthogonal nervous system) as plesiomorphies and not asapomorphies, and its peculiar duet spiral cleavage as a likelyplesiomorphic condition for Spiralia not necessarily derivedfrom the predominant quartet spiral modality. However, ex-clusion of various protostomes (e.g., most “Aschelminthes”)and all deuterostomes from his analyses (the branching ofCatenulida after the Rhabditophora, which is opposite to allinternal phylogenetic schemes for Platyhelminthes; Karling1974; Ax 1985, 1996; Ehlers 1985) were serious hindrancesagainst the basal position and the paraphyly of the group.

In conclusion, cladistic analyses have not unambiguouslyestablished the monophyly of the Platyhelminthes or theirlikely sister group. Because both questions hinge one uponanother and are one of the key points to settling the phylog-eny of the Bilateria and to exploring the transition fromradial to bilateral forms, the quality of currently availablemorphological cladistic data for the Platyhelminthes shouldbe reassessed and new characters called forward and ana-lyzed.

The importance of phylogenetic studies forthe evolution of parasitism and terrestriallife

The phylum Platyhelminthes comprises free-living andparasitic forms. Free-living forms are estimated between 4and 5000 species, whereas the parasitic forms, which makeup the majority of the Platyhelminthes, range from a low es-timate around 10 000 species to a high estimate around50 000 – 100 000 species. Such prevalence has attracted agreat deal of attention from biologists. However, they weremainly interested in the health and epidemiological aspectsof the impact of Platyhelminthes, leaving little room todwell on systematic issues. Early studies have taken a phe-netic approach, giving rise to a wide variety of evolutionaryscenarios on the origin and radiation of parasitic forms;however, none of which have been tested in meaningful orobjective ways. As with the phylogeny of Platyhelminthes asa whole, the introduction of cladistic studies on parasitic

Platyhelminthes have led to a number of clearer bifurcatingphylogenies (Ehlers 1985; Brooks 1989; Rohde 1990;Brooks and McLennan 1993). However, the characters onwhich these alternatives were drawn from were contentious,leaving a number of alternatives (Rohde 1990) and no singlerobust solution.

Despite these uncertainties, it is now well accepted thatthe parasitic Platyhelminthes, composed of the obligate para-sitic platyhelminth groups Monogenea, Trematoda, andCestoda, are a monophyletic group that, as first proposed byEhlers (1985), is known today as the Neodermata. However,a key point in understanding the origin and evolution of theNeodermata is to identify, within the free-living Platyhel-minthes, its sister group. In most cladistic analyses, the mostparsimonious solution found was an origin from groups thatwere commensals, ectoparasites, or obligate parasites (e.g.,“dallyeloid” rhabdocoels, which included Temnocephalida,Fecampiidae, and Udonellidae; Ehlers 1985); from varia-tions on the former scheme (Brooks 1989; Brooks andMcLennan 1993); or from a group of obligate parasites(Fecampiida and Urastomida; Joffe and Kornakova 1998)sharing a set of morphological synapomorphies with theNeodermata.

With an accurate phylogeny in hand, cladistic techniquescould be used for ancestral character reconstruction to pre-dict the most likely state of a character in any position of thetree. Therefore, finding the systematic position of the Platy-helminthes within the Bilateria and, further, finding thelikely sister group of the Neodermata within the Platyhel-minthes, would be extremely useful in making better in-formed guesses as to the attributes of parasitic taxa, inunderstanding how they could have evolved from free-livinggroups, and in developing better strategies and treatments tocontrol and eradicate them.

The transition from free-living marine or freshwater formsto free-living terrestrial form has been much less frequentthan the shift from free-living to parasitic forms. Even so,some Lecithoepitheliata (e.g., Prorhynchidae), some Rhab-docoela (e.g., Provorticidae), and, namely, the terrestrialTricladida (the Terricola, comprising more than 800 species)are groups that successfully colonized the land. The numberof studies devoted to physiological adaptations to terrestriallife in the Platyhelminthes is very sparse. Besides, its taxo-nomic and phylogenetic positions are not well understood.As for the Neodermata, knowledge of the sister group to theland Tricladida would be instrumental in making some edu-cated guesses as to whether terrestrial life evolved from ma-rine or freshwater ancestors. Moreover, it may give insightsas to which sort of preadaptations eased the way in the adop-tion of this life style that is at odds with some features of thePlatyhelminthes (e.g., lack of a hard, tough cuticle and norespiratory and circulatory systems).

The advent of molecular phylogeny: Thenew metazoan tree based on 18S ribosomalDNA (rDNA) sequences

The advantages of molecular sequences as phylogeneticcharacters was first shown by Woese et al. (1978) to inferthe first comprehensive phylogeny of prokaryotes using the16S ribosomal RNA (rRNA) subunit. This success prompted

© 2004 NRC Canada

172 Can. J. Zool. Vol. 82, 2004

the extension of this approach to eukaryotes, metazoansamong them, by sequencing their 18S rRNA subunit. Themain reasons for using 18S rDNA or rRNA have been re-peatedly reviewed (Woese 1987; Sogin 1991; Littlewoodand Olson 2001). The first contribution to metazoan phylog-eny using this approach was the seminal paper of Field et al.(1988). However, early works using 18S rDNA or othermolecules were affected by a set of similar problems:(i) limited or very limited number of sampled taxa; (ii) se-lection of crown instead of basal species; (iii) insufficientnumber of characters owing to the use of partial sequences;(iv) unequal rates of molecular evolution for different taxagiving rise to the “Long Branch Attraction” problem (LBA;Felsenstein 1978); (v) rate heterogeneity across sites of thesame molecule creating conflicting phylogenetic signalswithin the same molecule; (vi) molecular compositional bi-ases; and (vii) mutational saturation at variable sites (satura-tion effect). Altogether, this led to the view that 18S rDNAdata could not be taken as the panacea for metazoan phylog-eny and evolution, especially to sort out its deepest nodes(Abouheif et al. 1998).

However, these problems were soon tackled and mendedusing different tests and analyses. The resulting full-length18S rDNA new molecular trees (Halanych et al. 1995;Aguinaldo et al. 1997; Adoutte et al. 1999, 2000) broughtimportant findings that strongly contested several basic ten-ets of morphologically based trees (Fig. 3A, 3B). First, and

besides the undisputed monophyly of metazoans as a whole,bilaterians appeared as a clearly separated group fromsponges, cnidarians, and ctenophores (the Diploblasts orRadialia). Within the Diploblasts, its emergence sequencewas left unresolved. Second, and in contrast to a two-century-old tradition, the “Articulata” (the clade of seg-mented organisms that grouped annelids and arthropods)were split, uniting annelids with several protostome phylathat share spiral cleavage and a trochophore larva (e.g.,molluscs, nemertines, echiurans, etc.). Third, the lophophor-ates (brachiopods, bryozoans, and phoronids) sharing a setof tentacles surrounding the mouth, or lophophore, andthought to be closer to the deuterostomates than to the proto-stomates, clearly affiliated with the later. Lophophorates,together with the group mentioned above which contain theannelids and molluscs, emerged together in a single super-clade that was named Lophotrochozoa (Halanych et al.1995). Such name is in need of revision as it incorporatestwo characters (the lophophore and a trochophora larva) notshared by all members of the clade. Fourth, the acoelomates(basically the Platyhelminthes and the Nemertea; Hyman 1951)were brought within the Lophotrochozoa by 18S rDNA.Fifth, the pseudocoelomate clades or “Aschelminthes”(sensu Hyman 1951) exploded and some groups (e.g.,Priapulida, Kinorhyncha, Nematomorpha, and Nematoda),together with arthropods and close groups (e.g., Tardigrada,Onychophora), emerged as a sister group to lophotro-

© 2004 NRC Canada

Baguñà and Riutort 173

Fig. 3. Comparison of alternative phylogenies for the Metazoa, with the traditional scheme based on morphological and embryologicalcharacters (adapted from Hyman 1951) (A) and a consensus molecular tree based on 18S ribosomal DNA (rDNA) sequence data andnumber and type of genes from the Hox cluster (adapted from Adoutte et al. 2000) (B). In both trees, Platyhelminthes are boxed. Notethe unresolved polytomies for Lophotrochozoa and Ecdysozoa in Fig. 3B. D, Deuterostomia; L, Lophophorates; P, Protostomia. Seetext for further details.

chozoans. Such group or superclade was named Ecdysozoa(Aguinaldo et al. 1997), as all members share the presence ofa moulting cuticle (albeit of different composition). In turn,other “Aschelminthes” groups (e.g., Rotifera, Gastrotricha)took uncertain positions either with lophotrochozoans orwith ecdysozoans. And finally, the deuterostomes, withoutthe lophophorates, appeared as a monophyletic group sisterto the “Protostomia”, now divided into Ecdysozoa and Lo-photrochozoa.

In summary, the 18S rDNA phylogeny clearly supportedthe existence of three basic superclades in the Bilateria:Deuterostomia, Ecdysozoa, and Lophotrochozoa. In addi-tion, acoelomate (Platyhelminthes among them) and pseudo-coelomate groups, once considered intermediate formsbetween diploblasts and bilaterians, were now displaced to amuch higher position inside the tree (Adoutte et al. 1999).Such scenario backed the archicoelomate (now renamed the“complex Urbilateria”) hypothesis (Kimmel 1996; DeRobertis 1997; Holland 1998; Adoutte et al. 1999) in frontof the classical planuloid–acoeloid hypothesis. However, aspointed out by Jenner (2000), most published phylogeniessupporting a “complex Urbilateria” were heavily pruned asthey left out several “minor” phyla (namely the basalecdysozoans and lophotrochozoans) to which most pseudo-coelomates and acoelomates belong. In addition, the pro-posed gene expression pattern homologies were unreliableand exhibited a strong taxon selection. Altogether this meantthat the emerging molecular view of animal evolution wasbased on incomplete and unresolved phylogenies.

Molecular phylogenetics of thePlatyhelminthes

Among 10 other different metazoan taxa, Field et al.(1988) included 1 single platyhelminth, the freshwatertriclad Girardia tigrina, that branched out as the first bilater-ian. Such result fitted the commonly held view of platy-helminths as early branching bilaterians. This position wasfurther corroborated when seven platyhelminths, represent-ing two free-living orders and one parasitic species, werealso included within a larger sample of metazoans (Riutort etal. 1992, 1993). However, as mentioned above, insufficienttaxon sampling and the use of partial 18S rRNA sequencesmade the resulting trees unreliable. Similar problemsplagued trees reported by Rohde et al. (1993, 1995),Jondelius (1998), Campos et al. (1998), and Litvaitis andRohde (1999). In all of them, placement of the Platyhel-minthes within the Metazoa was not addressed or resulted inodd positioning (i.e., the internal phylogeny was mostly atodds with the Karling (1974) and Ehlers (1985) proposalsbased on morphological characters).

Platyhelminthes are not monophyleticThe first comprehensive molecular tree for the Platyhel-

minthes based on complete 18S rDNA sequences was pub-lished by Carranza et al. (1997). It included 16 speciesbelonging to several free-living orders and some parasiticclasses, and used different methods of phylogenetic infer-ence. Its main aim was to test the tenet that Platyhelminthesare a monophyletic group of basal bilaterians which form the

sister group to other bilaterian phyla. Surprisingly, Platy-helminthes were either polyphyletic or paraphyletic withAcoela and Catenulida, branching out in sequence as thefirst bilaterians, whereas the majority of the Platyhelminthes,the Rhabditophora, branched within the Protostomia. Usinga much more limited number of species, the deep position ofthe Rhabditophora was also reported by Balavoine (1997),whereas a single species of Catenulida clustered at the baseof a new superclade, the Lophotrochozoa (sensu Halanych etal. 1995), which together with the Ecdysozoa divided theProtostomia (Aguinaldo et al. 1997).

However, the paper by Carranza et al. (1997) containedseveral anomalies, which were new and interesting. First,tree topology and the polyphyletic or paraphyletic nature ofPlatyhelminthes varied depending on the method of infer-ence used. Second, the position of Catenulida outside thePlatyhelminthes and only second to acoels at the base of theBilateria was not well supported. Third, although Acoela ap-peared with moderate support as the first bilaterians, theirsequences showed a great number of differences when com-pared with any of the organisms included in that study. Thus,they behaved as fast-clock organisms and under such situa-tions were suspected of causing LBA artifacts (Felsenstein1978). Such features led the authors to skip acoels from therest of the analyses and to call for new data to establish theposition of acoels. Finally, and oddly enough, the Nemer-todermatida, which were considered on the basis of goodmorphological synapomorphies to be the sister group of theAcoela and forming the clade Acoelomorpha (Ehlers 1985),were grouped inside the rest of the Platyhelminthes (theRhabditophora) within the Lophotrochozoa, and separatelyfrom the acoels.

The Acoela: Platyhelminthes or basal bilaterians?The position of Acoela as basal bilaterians had previously

been claimed from partial 18S rRNA sequences. Katayamaet al. (1993) found Platyhelminthes as the earliest bilateriansforming a paraphyletic group, with acoels the earliest emerg-ing bilaterian clade, followed by Tricladida, Polycladida,Nematoda, the arthropod Artemia sp., and three Chordata. Asimilar position resulted from a later, wider sampling work,which included the same two acoel species (Katayama et al.1996). However, both reports had most of the flaws of early18S phylogenetic studies: use of partial sequences; a limitedrange of representatives from the rest of the metazoans; anda too distant outgroup (the fungi), which enhanced the LBAeffects. Such effects and the likely artefactual position ofacoels were also present in later metazoan wider sampling(Campos et al. 1998; Zrzavy et al. 1998; Littlewood et al.1999a; and Giribet et al. 2000), namely because the same setof acoels (Campos et al. 1998; Zrzavy et al. 1998; Giribet etal. 2000) or new species, still long-branched, were used(Littlewood et al. 1999a), or because solely long-branchedgroups (e.g., gnathostomulids, nematodes, acanthocephalans)were included, whereas representatives from some key meta-zoan groups (e.g., deuterostomes in Campos et al. 1998 andLittlewood et al. 1999a) were not. Surprisingly, in sometrees (Campos et al. 1998; Giribet et al. 2000), acoels didnot branch as basal bilaterians but deep within the Rhab-ditophora, forming the sister group of the Tricladida. These

© 2004 NRC Canada

174 Can. J. Zool. Vol. 82, 2004

results were a likely consequence of LBA because mosttriclads were also fast-clock organisms. Moreover, a numberof morphological synapomorphies present in Tricladida andother Rhabditophora, but missing in the Acoela (e.g., hetero-cellular female gonads, rhabdites, protonephridia), conflictedwith a position of Acoela close to Tricladida within theRhabditophora.

A way out of such a standstill was to obtain 18S rDNAsequences of more Acoela species to find one (some) thatdid not show the fast-clock behaviour of the rest. Complete18S rDNA sequencing of 18 species of acoels enabled Ruiz-Trillo et al. (1999) to identify one species, Paratomellarubra, as having evolved at a sufficiently slow rate that LBAeffects may be avoided. Ruiz-Trillo et al. (1999) performed arelative rate test to ascertain that only taxa with similar sub-stitution rates were included in the analysis. In a relative ratetest, species are compared by pairs to test if their distance tothe outgroup is significantly different. If the distance is sig-nificantly different, then one of them has a significantlyhigher rate of substitution than the other. In such analyses,species with significantly higher rates of evolution to mostother species could be considered a fast-clock species, andhence prone to LBA effects in the phylogenetic inference.Of 74 bilaterian species tested in Ruiz-Trillo et al. (1999),57 (representing 21 phyla) passed the test. The resulting tree(under maximum-likelihood analysis) gave three monophy-letic superclades: Deuterostomia, Ecdysozoa, and Lophotro-chozoa, with acoels as the first offshoot after the diploblasts.Moreover, the majority of the Platyhelminthes, the Rhab-

ditophora, and the Catenulida clustered within, but at thebase of, the Lophotrochozoa (Fig. 4). The use of only nonfast-clock sequences and of a number of tests to avoid LBAartifacts ensured the goodness of the result.

The proposal of acoels as a basal bilaterian clade and notbelonging to the Platyhelminthes was soon contested. First,phylogenetic analysis of amino-acid sequences of Elonga-tion Factor 1-alpha (EF1-alpha) contradicted such a position(Berney et al. 2000). However, reanalysis of this and newdata led Littlewood et al. (2001a) to the conclusion thatBerney et al.’s proposal had some methodological flaws and,when considered in isolation, EF1-alpha sequences con-tained insufficient signal for a reliable placement of acoels.Second, Giribet et al. (2000) and Peterson and Eernisse(2001) claimed that inspite of running several tests to avoidLBA effects the branch length of the single acoel included inthe Ruiz-Trillo et al. (1999) tree may have actually been “at-tracted” to the long branch separating non-bilaterians (diplo-blasts) and bilaterians, and they (Giribet et al. 2000)advocated the skipping of the diploblasts as outgroups. Suchcriticisms are not fully justified. The only acoel in Ruiz-Trillo et al. (1999), P. rubra, passed a relative rate test; incontrast, Giribet et al. (2000) used two species that were nottested and were actually fast-clock species, and they used aninference method (i.e., maximum parsimony) that was moreprone to LBA. In turn, Peterson and Eernisse (2001) in-cluded three acoels that were rejected in the relative rate testby Ruiz-Trillo et al. (1999). Finally, the major contentionagainst acoels as basal bilaterians resulted from the odd po-

© 2004 NRC Canada

Baguñà and Riutort 175

Fig. 4. Diagram of the best 18S-rDNA-based maximum-likelihood tree of 61 metazoan species with homogeneous rates of nucleotidesubstitution illustrating the basal position of the Acoela (set in boldfaced and uppercased type) within the Bilateria and the position ofthe rest of the Platyhelminthes (PL), which falls within the superclade Lophotrochozoa. This renders the Platyhelminthes polyphyletic.Note the position of the order Nemertodermatida (nemertod set in boldfaced type) buried within the Platyhelminthes. Scale bar repre-sents the number of nucleotide substitution per site. The number 100 on the branch separating acoels from the rest of bilaterians repre-sents the percent support of that branch by the four-cluster likelihood mapping (modified from Ruiz-Trillo et al. 1999). See text formore details.

sition of the representative of the other acoelomorph group(Nemertinoides elongatus, order Nemertodermatida), whichin Ruiz-Trillo et al. (1999) grouped unambiguously with thePlatyhelminthes. Because acoels and nemertodermatidsshared robust morphological characters (ciliary rootlet sys-tem, bent cilia, and likely, duet-type spiral cleavage), mostzoologists believed that the placement of acoels was proba-bly erroneous and preferred the position of the Nemerto-dermatida within the rhabditophoran Platyhelminthes, whichwas also indicative of the acoel relationship (Adoutte et al.2000; Peterson et al. 2000; Dewel 2000; Telford 2001).However, complete 18S rDNA sequences from three addi-tional species of nemertodermatids unambiguously showedthem to cluster with acoels at the base of the bilaterians andnot with the rhabditophoran Platyhelminthes (Jondelius et al.2002). This result also implied that the sequence of N. elon-gatus from Carranza et al. (1997) and used in Ruiz-Trilloet al. (1999) and in all analyses therein was an unfortunatesequencing artefact or originated from an undisclosedrhabditophoran flatworm.

In conclusion, all molecular evidence brought forwardfrom complete 18S rDNA sequences supported, after under-taking the right tests and analyses, the position of acoels andnemertodermatids as the extant earliest bilaterians and theplacement of the Platyhelminthes within the Lophotrochozoa.The position of acoelomorphs as the earliest extant bilaterianmetazoans have deep implications for the phylogeny andtaxonomy of Platyhelminthes and for the origin and evolu-tion of bilaterans. First, it strongly supports the polyphyly ofPlatyhelminthes and the need to consider Acoelomorpha as aseparate phylum. Second, it implies that the last commonbilaterian ancestor was small, benthic, without segments andcoelomic cavities, and likely with direct development. Theselend support to the planuloid–acoeloid hypothesis of bila-terian origins and argue against a “complex Urbilateria” asthe earliest bilaterian. Finally, it also argues for an extendedperiod before the Cambrian within which different bilaterianlineages may have originated, with the acoels being the de-scendants of one of these lineages.

Further characters supporting a basal position for theAcoelomorpha and the polyphyly of thePlatyhelminthes

The basal position of the Acoelomorpha and the ensuingpolyphyly of Platyhelminthes have been further tested andcorroborated using new molecular and embryological char-acters. First, the most comprehensive analyses of genes ofthe cluster Hox from different rhabditophoran Platyhelmin-thes, namely triclads and polyclads (Balavoine 1998;Bayascas et al. 1998; de Rosa et al. 1999; Orii et al. 1999;Nogi and Watanabe 2001; Saló et al. 2001), have clearlyshown the presence of an almost full set (7–8 genes) of Hoxgenes. Moreover, some genes bear the so-called “signature”peptides outside the homeobox (e.g., the spiralian peptide or“Lox5 peptide” and the “UbdA peptide”; Balavoine 1998; deRosa et al. 1999), which should be very good indicators oflophotrochozoan and “protostome” relationships, respectively.The discovery of such molecular signatures in acoel Hoxgenes has been mentioned (as “unpublished work” inAdoutte et al. 1999) but never been substantiated. Instead,

recent work suggest that the acoels Symsagitiffera roscoffen-sis and P. rubra do have a limited set of Hox genes (4 to 5)that do not bear neither lophotrochozoan nor ecdysozoanmolecular signatures (Cook et al. 2004). Secondly, Telfordet al. (2000) checked the presence of rare changes in mito-chondrial genetic code characteristics of the Platyhelminthes.Two such changes were present in all rhabditophorans ana-lyzed but not in catenulids, acoels, and nemertodermatids.Although this does not necessarily support the polyphyly ofthe Platyhelminthes, it proves that none of these groups havebeen derived from within the Rhabditophora (opposite of theresults of Campos et al. (1998), Giribet et al. (2000), andBerney et al. (2000) for acoels, although see Giribet (2002)).Thirdly, sequences from the myosin heavy chain type II(myosin II) gene from a large set of metazoans, includingacoels and nemertodermatids, have demonstrated for the my-osin II data set alone and for a combined 18S rDNA + myo-sin II data set the polyphyly of Platyhelminthes and thebasal position of Acoelomorpha (Ruiz-Trillo et al. 2002).Fourth, a recent combined data set of 18S + 28S rDNA genesequences have shown again that Acoelomorpha are themost basal known triploblastic Bilateria (Telford et al.2003). And fifth, the heterochronic gene let-7 from the nem-atode Caenorhabditis elegans, an essential regulator of de-velopmental timing, present consistently in samples from allbilaterians (including triclad and policlad rhabditophoranplatyhelminths) but absent from all diploblasts tested, hasnot been found in acoels (Pasquinelli et al. 2003). Additionalmolecular data, although still preliminary, are available fromthe very different mitochondrial gene arrangements betweenacoelomorphs and rhabditophoran Platyhelminthes (Ruiz-Trillo et al. 2004).

Together with genes and molecules, some morphologicalcharacters also lend support to the basal position of acoelo-morphs and the polyphyly of Platyhelminthes. Embryoniccell cleavage in rhabditophorans, exemplified by polyclads(Boyer et al. 1996, 1998), follows a stereotypic quartet spiralpattern strikingly similar to that of spiralian lophotrocho-zoans (e.g., annelids, molluscs, etc.). Moreover, cell-lineageanalysis also show, despite unique aspects for polyclads, aremarkable similarity with those of higher spiralian em-bryos, including derivation of mesoderm from both ecto-dermal (2b cell) and endodermal precursors (4d cell) (Boyeret al. 1998). In contrast, acoel embryonic cleavage and celllineage (Henry et al. 2000) are of the duet spiral type andthe endomesoderm is the sole source of mesoderm. As dis-cussed by Henry et al. (2000), duet cleavage have very dis-tinct features compared with the typical quartet cleavage: itis actually more bilateral than spiral; cleavages do not alter-nate clockwise and anticlockwise; and blastomere fates aredifferent to those of quartet spiral embryos. The duet cleav-age has always been considered derived from the quartettype (Ax 1987, 1996; Ax and Dörjes 1966; for the oppositeview see Haszprunar 1996a). However, given the differencesbetween both types and the new position of acoels, duet spi-ral cleavage could have arisen from a form of radial orbiradial cleavage characteristic of the more primitive cleav-age programs in the Metazoa, whereas quartet spiral cleav-age could have had an independent origin within thelophotrochozoan clade. A similar argument could be raisedfor another acoelomorph feature, the lack of protonephridia.

© 2004 NRC Canada

176 Can. J. Zool. Vol. 82, 2004

© 2004 NRC Canada

Baguñà and Riutort 177

In most schemes (Karling 1974; Ehlers 1985; Ax 1996; butfor a different view see Haszprunar 1996a), it is regarded asan apomorphy, deviating from the ground plan of the Platy-helminthes. However, lack of protonephridia may be a sym-plesiomorphy linking acoelomorphs to diploblasts instead ofto catenulids and rhabditophoran Platyhelminthes. Finally,the presence in acoelomorphs of an anterior concentration ofnerve cells without forming a “true brain” (with neuropile)(Reuter et al. 1998; Raikova et al. 2000; but for an oppositeview see Tyler 2001), together with the lack of asymmetri-cally distributed longitudinal nerve cords (either dorsal orventral), may also indicate that they are not related toPlatyhelminthes.

In summary, molecular and morphological evidencebrought forward show that the Acoelomorpha are the mostbasal known triploblastic Bilateria and the Platyhelminthesare a polyphyletic clade. Awaiting new corroborative evi-dence, Bilateria could be divided in two inclusive groups: abroad Bilateria including acoelomorphs and a more derivedBilateria, or Eubilateria, excluding this clade (Fig. 5). Syna-pomorphies for all bilaterians would be the two orthogonalbody axes, anterior nervous system, and endo-mesoderm.Eubilateria could be defined by the presence of an excretorysystem, one-way through gut, further development of thenervous system, and a full complement of Hox cluster genes.If this scheme holds true, then Acoelomorpha should be es-tablished as a new phylum as first pointed out by Ruiz-Trillo

et al. (1999) from their analysis of 18S rDNA trees. Besides,the planula-like features of acoels and nemertodermatidssupport again the planuloid–acoeloid hypothesis of bilaterianorigins and draw attention to features of the embryonic de-velopment of acoelomorphs as a way to explore howbilaterians originated and evolved.

The position of the Catenulida and the Rhabditophorawithin the Lophotrochozoa

The position of the Catenulida within Platyhelminthes andof other bilaterians in the first complete 18S rDNA trees(Carranza et al. 1997; Zrzavy et al. 1998; Littlewood et al.1999a; Giribet et al. 2000) was uncertain because of insuffi-cient taxon sampling and (or) inclusion of taxa (e.g.,Gnathostomulida, Nematoda, Acanthocephala, Acoela,among others) with high or very high rates of nucleotidesubstitution. When such problems were taken into account(Ruiz-Trillo et al. 1999), Catenulida appeared as the sistergroup to the Rhabditophora and within the Lophotrochozoa.Recent, denser sampling works using 18S rDNA data(Baguñà et al. 2001; Peterson and Eernisse 2001; Jondeliuset al. 2002) or combined 18S + 28S rDNA data sets (Telfordet al. 2003) strongly support this sister-group relationship.Such relationship is reinforced by the lack in the Catenulidaof the synapomorphic rhabditophoran mitochondrial geneticcode changes (Telford et al. 2000). However, it is importantto bear in mind that Smith et al. (1986) did not find strong

Fig. 5. A new systematic and phylogenetic proposal for the Metazoa based on molecular (18S + 28S rDNA, Hox cluster genes, myosinII, and let-7 gene data) and morphological characters. The Bilateria is divided into acoels and nemertodermatids, which form theAcoelomorpha, and the rest of the bilaterians, or Eubilateria, which itself divide into the three large superclades Deuterostomia,Ecdysozoa, and Lophotrochozoa. Note the position of the majority of the Platyhelminthes within the Lophotrochozoa, and the former“Aschelminthes” (or pseudocoelomates) now divided within the Ecdysozoa and the Lophotrochozoa. Bilaterian autapomorphies are asfollows: BIL, bilaterial symmetry with two body axes (anteroposterior or AP and dorsoventral or DV); END, endomesoderm; andANS, anterior nervous system. The Eubilateria will have some autapomorphies that exclude the acoelomorphs and are as follows: BG,fully formed brain ganglia; ES, excretory system; and G, one-way gut (mouth + anus). Synapomorphies uniting acoels andnemertodermatids into the Acoelomorpha are as follows: DC, duet spiral cleavage; CS, interconnecting ciliary root system; and BC,bent cilia at terminal ends. In turn, acoels and nemertodermatids have a particular set of autapomorphies, which are as follows: 1,statocist with one statolith and two parietal cells; 1′, statocist with two statoliths and several parietal cells; 2, absence of extracellularmatrix; 3, absence of gut glandular cells; and 4, biciliary sperm. See text for further details and main references.

© 2004 NRC Canada

178 Can. J. Zool. Vol. 82, 2004

morphological synapomorphies uniting Catenulida andRhabditophora.

Which is the bilaterian or, better yet, the lophotrochozoansister group to the Catenulida + Rhabditophora? So far nei-ther morphology nor molecules alone, and from total-evidence analyses of 18S rDNA sequence data and morphol-ogy, have provided a clear answer. Zrzavy et al. (1998) stillplaced Catenulida and Rhabditophora paraphyletically at thebase of the Bilateria, whereas Giribet et al. (2000) andZrzavy (2001) placed them within a platyzoan grade(Platyzoa after Cavalier-Smith 1998) within the Protostomia(Giribet et al. 2000) or in an unresolved position (Zrzavy2001). After combining morphology and 18S rDNA analy-ses, including or not long-branched organisms (acoels,gnathostomulids, or gastrotrichs), Peterson and Eernisse(2001) found Catenulida + Rhabditophora to be a mono-phyletic group at the base of the Lophotrochozoa, sistergroup to the Lophophorates + Trochozoa. Such basal posi-tion, second only to Gastrotricha, was also reported in Ruiz-Trillo et al. (1999) and Jondelius et al. (2002).

In conclusion, molecular sequence data tentatively sug-gests that Catenulida + Rhabditophora is a monophyleticclade close to the base of the Lophotrochozoa. However, theuncertain positions of gastrotrichs, gnathostomulids, acan-tochephalans + rotifers, and most lophophorates togetherwith the extant poorly resolved internal phylogeny of lopho-trochozoans (Fig. 3B) call for a denser sampling of basal,non-fast evolving species from such clades. A striking con-clusion is that, despite current views of Platyhelminthes (tothe exclusion of the Acoelomorpha) as an acoelomate groupderived from coelomate ancestors (Smith and Tyler 1985;Rieger 1986; Balavoine 1998; Adoutte et al. 1999, 2000),their likely placement at the base of the Lophotrochozoamakes necessary the reassessment of the phylogenetic signif-icance of several state characters (e.g., reduction of thehindgut and absence of anus, lack of coelom, and larva withreduced hyposphere) taken as evidence of its secondarily de-rived condition. Similar arguments could be raised for theacoelomate Gastrotricha if its new suggested basal positionas sister group to the rest of the Ecdysozoa holds true(Zrzavy 2003).

The internal phylogeny of thePlatyhelminthes based on 18S rDNA trees

Towards a consensus tree?Morphological assessments of interrelationships of Platy-

helminthes are, since the rigorous early efforts of Karling(1974) and Ehlers (1985) and the subsequent works of Smithet al. (1986), Ax (1996), Haszprunar (1996a, 1996b), andLittlewood et al. (1999a) among others, based on interpreta-tions of character homology. Although these works laid torest the old taxonomy of the turbellarian Platyhelminthes

based on features of the gut system (Acoela, Alloeocoela,Rhabdocoela, Tricladida, and Polycladida; Hyman 1951),the only consensus emerging from it (see Figs. 2A–2C) wasthe recognition of three clear monophyletic groups: Caten-ulida, Acoelomorpha, and Rhabditophora (Table 1). In somereports these three clades were left unconnected (Smith et al.1986; Fig. 2C) or made paraphyletic (Haszprunar 1996a;Fig. 2D), whereas the branching order among them differedsubstantially (e.g., compare Figs. 2A and 2B with Fig. 2D).Within the Rhabditophora, the branching order usually hadthe Macrostomida and the Polycladida at the base, with thederived groups such as Rhabdocoela, Prolecithophora, andTricladida at the crown and the parasitic classes (theNeodermata; Ehlers 1985) usually stemming from theRhabdocoela (however, for a substantially different orderingsee Littlewood et al. 1999a). Other minor groups, such asthe Lecithoepitheliata and the Haplopharyngida, clustered ei-ther at the base or at the crown.

As with the position and the monophyly/polyphyly of thePlatyhelminthes, molecular data (namely 18S rDNA gene se-quences) have furnished an independent source of phylogen-etic information regarding the internal phylogeny of the group.The attempts made since the early 1990s have been aptlysummarized by Littlewood and Olson (2001) and, especiallythose using partial sequences, will not be further reviewedhere. Katayama et al. (1996) provided the first comprehen-sive tree (Fig. 6A), including several orders, based on com-plete 18S rDNA sequences. Because most long-branchedorganisms were not trimmed and other bilaterians were notincluded, the resulting tree had several anomalies: acoelswere most basal, followed by long-branched triclads thatwere the sister group to all other flatworms. Moreover, cate-nulids appeared buried within the Rhabditophora, polycladswere not basal, and in all trees, parasitic classes formed amonophyletic clade sister group to the rhabdocoels. Soon af-ter, Carranza et al. (1997) produced the first tree (Fig. 6B),close to what is today accepted as a “consensus” tree (seebelow). They avoided LBA problems by leaving the acoelsout of the tree. The resulting tree had macrostomids andpolyclads at the base of the Rhabditophora, followed by alarge group formed by a paraphyletic Seriata (Tricladida +Proseriata) clustering with Lecithoepitheliata, Prolecitho-phorata, and Rhabdocoelsa, and, strikingly, altogether sistergroup to a clade made by the parasitic classes (theNeodermata). The tree in Fig. 6B, however, had two anoma-lies. First, the paraphyletic position of Catenulida to the restof the Platyhelminthes. Second, as already discussed, theartifactual position of the acoelomorph N. elongatus(Nemertodermatida) within the Rhabditophora, which is ananomaly that plagued other studies. In addition, taxon sam-pling was not wide enough to make some nodes statisticallyreliable.

The phylum-wide embracing study by Littlewood et al.

Fig. 6. Recent phylogenetic proposals on the interrelationships of the Platyhelminthes based on 18S rDNA sequence data (for currentclassification of the Platyhelminthes see Table 1). Open triangles represent monophyletic complex groups (e.g., Neodermata,Rhabdocoela) or non-monophyletic unresolved clades. Dotted lines indicate uncertain relationships. Trees from Katayama et al. (1996)(A), Carranza et al. (1997) (B), Littlewood et al. (1999a) (C), Littlewood and Olson (2001) (D), Baguñà et al. (2001) (E), and a con-sensus tree (F). Note that trees in Figs. 6A, 6B, and 6C have been redrawn from Littlewood and Olson (2001), whereas the othershave been adapted from the references indicated. See text for further details.

(1999a) used 82 sequences of complete 18S rDNA and in-corporated 65 morphological characters mainly drawn fromthe Rohde (1990) matrix to produce the first combined anal-ysis on Platyhelminthes (Fig. 6C). Molecular trees reflected

the main features of Carranza et al. (1997), with some im-portant improvements such as catenulids being the mostbasal group, and Lecithoepitheliata and Haplopharyngida re-situated close to polyclads and macrostomids, respectively.

© 2004 NRC Canada

Baguñà and Riutort 179

More importantly, the paraphyly of the Seriata was corrobo-rated, and the Neodermata position as sister group to a largeclade including Rhabdocoela and Tricladida was also cor-roborated and refined. Morphologically based trees con-flicted with molecular analyses. Whereas Neodermata wasshown to be a monophyletic sister group to a large clade ofrhabditophorans and other “reasonable” groupings were alsoreproduced, polytomies (under strict consensus) or oddgroupings (e.g., triclads with polyclads; prolecithophoranswith lecithoepitheliates) under a 50% majority rule consen-sus plagued the trees. Total-evidence trees under maximumparsimony were almost identical to that given by maximum-parsimony analysis of the 18S rDNA alone; not surprisingly,morphology and molecular data were incongruent but in-compatible under Templeton’s test. Later, Littlewood et al.(1999b) added a few more taxa (up to 97) for 18S rDNA se-quences, included partial 28S rRNA sequences (D1 and D3–D6 domains), and used the same morphological matrix.Where it had previously failed (Littlewood et al. 1999a),18S rDNA and morphology data sets were now incongruentbut compatible under Templeton’s test. Instead, 28S rRNAwere incompatible with 18S rDNA and morphology datasets, likely because sampling was inadequate. However,Neodermata was shown again to be a monophyletic groupand sister group to a large clade of neoophoran rhabdito-phorans supporting, as already pointed out by Carranza et al.(1997), an early emergence of parasitic Platyhelminthes (seebelow).

A comprehensive effort to find a consensus internal treefor the Platyhelminthes using 18S rDNA data sets was pub-lished by Littlewood and Olson (2001). Using minimum-evolution and maximum-parsimony approaches, they used270 taxa (180 parasitic species and 90 free-living species) ina concentrated effort to find the main relationships betweenthe major clades. Rate-site heterogeneity, effects of the sec-ondary structure (stem and loops), and mutational saturationwere taken into account in the analyses. The resulting tree(Fig. 6D; see Figs. 25.5 and 25.8 in Littlewood and Olson2001) with the Catenulida as the outgroup confirmed themonophyly of the Macrostomida and Haplopharyngida,Polycladida, Lecithoepitheliata, Tricladida, Prolecithophora,Rhabdocoela, Neodermata, and of a clade comprising theparasitic “turbellarian” genera Icthyophagha, Notentera,Urastoma, and Kronborgia. Neodermata was the sister groupto a clade composed of Proseriata, Rhabdocoela, Fecampiida(+ Urastomidae), Prolecithophora, and Tricladida. Relation-ships among the earliest divergent Platyhelminthes were,however, not well resolved with Polycladida, Lecithoepithe-liata, Macrostomida, and Haplopharyngida, which formedgroups with low and variable support in maximum-

parsimony and minimum-evolution trees. A similar tree wasreported by Baguñà et al. (2001) using 72 Platyhelminthes(including 5 parasitic taxa) rooted to other bilaterians(Fig. 6E) under neighbor-joining and maximum-likelihoodanalyses. Catenulids appeared highly supported at the base,followed by a poorly supported sequence of monophyleticMacrostomida + Haplopharyngida, Polycladida, and Lecito-epitheliata. Interestingly, Neodermata was shown to be amonophyletic clade, sister group to Rhabdocoela, Fecampi-idae (+ Urastomidae), Prolecithophora, and Tricladida. Thisexcluded the Proseriata that fell in a more basal position assister-group of the clade formed by Neodermata and thesederived rhabditophorans.

A last attempt to reach a consensus tree has been made byLockyer et al. (2003) combining nearly complete sequencesof 18S and 28S rDNA from 32 clades of Platyhelminthesrooted against the Catenulida using maximum-parsimony,maximum-likelihood, and Bayesian-inference analyeses. Atvariance with the former analysis of Littlewood et al.(1999b), all methods provided congruent estimates of phy-logeny. However, short internal branches and polytomies stillaffected the most basal clade formed by polyclads, macro-stomids, and lecithoepitheliates. Interestingly, Neodermatawas shown to be the sister group of a clade of derivedrhabditophorans to the exclusion of the Proseriata, whichwas more basal as suggested in Baguñà et al. (2001). 28SrDNA sequence data alone provided for all methods poorresolution at deeper nodes. Maximum-parsimony analyseswere, as in Litvaitis and Rohde (1999), especially confusingbecause of odd clusterings (polyclads and macrostomids assister group to Neodermata), although some stretches of the28S rDNA appeared useful in resolving higher nodes. Com-bined and complete 18S + 28S sequence data supported ear-lier studies based on 18S rDNA sequences alone (Baguñàet al. 2001; Littlewood et al. 2001b). The most basalRhabditophora is a clade made by the Macrostomida (orMacrostomorpha; Rieger 2001), Polycladida, and Lecitho-epitheliata. Of the remaining orders, a monophyletic Pro-seriata branches first, sister group to a clade made by theNeodermata and other turbellarians: Rhabdocoela, Tricladida,Prolecitophora, and a group made by the genera Ichthy-ophaga (Genostomatidae), Notentera (Notonteria), Urastoma(Urastomidae), and Kronborgia (Fecampiidae) (or INUK).

Although sampling of the Catenulida and several rhab-ditophoran groups (namely Macrostomorpha, Polycladida,Lecitoepitheliata, Proseriata, and some Rhabdocoela fami-lies) needs to be expanded, the tree shown in Fig. 6F, mainlybased on 18S rDNA sequence data, represent the best avail-able consensus molecular tree of the Platyhelminthes today.However, such a tree should be corroborated by adding new

© 2004 NRC Canada

180 Can. J. Zool. Vol. 82, 2004

Fig. 7. Phylogenetic trees depicting various proposals for the internal phylogeny of the Neodermata and its relationships to the rest ofthe Platyhelminthes (for current classification of the Platyhelminthes see Table 1). C, Cestoda; M, Monogenea; and T, Trematoda.Short horizontal black bars (Neodermata) indicate the origin of obligate parasitism. Revertospermata indicates the taxon unitingPlatyhelminthes with neodermatan-type spermiogenesis as proposed by Kornakova and Joffe (1999). Trees after Ehlers (1985) andbased on morphological characters (A); after Brooks and McLennan (1993) and based on morphology (B); the revertospermata pro-posal after Kornakova and Joffe (1999) (C); consensus most parsimonious tree combining 18S rDNA and morphology after Littlewoodet al. (1999b) (D); maximum-parsimony solution for 270 complete 18S rDNA gene sequences adapted from Littlewood and Olson(2001) (E); and consensus tree based on 18S + 28S rDNA data sets using maximum-likelihood and Bayesian-inference analyses fromLockyer et al. (2003) (F). See text for further details.

© 2004 NRC Canada

Baguñà and Riutort 181

data, namely sequences of new genes and (or) gene or ge-nome features that could represent molecular synapomor-phies. In addition, total-evidence analysis should makecongruent molecular trees with morphological trees providedthat morphological matrices were previously reassessed to

avoid uncertain characters as well as to incorporate newones.

The sister group to the NeodermataThe literature on the origin of parasitism in Platy-

helminthes and on the nature of its sister group is reviewedin Littlewood et al. (1999a, 2001b), resulting in three mostplausible scenarios. The first scenario (Fig. 7A) followsEhlers (1985) arguments for a clade of “dallyeloid” rhab-docoels that include the Temnocephalida, Fecampiidae, andUdonellidae, which are commensals, ectoparasites, or obli-gate parasites of several crustaceans, respectively. The sec-ond scenario stems from the proposal of Brooks (1989) andBrooks and McLennan (1993) of a Cercomeria superclassformed by a clade that includes Temnocephalida and its sis-ter group Neodermata + Udonellida (Fig. 7B). The third sce-nario is the proposal from Joffe and Kornakova (1998) of aunited clade called Revertospermata formed by Fecampiida,Urastomidae, and Neodermata (Fig. 7C). The last proposal issupported by strong morphological evidence from spermmorphology and spermatogenesis and by the fact that allmembers of the Revertospermata are obligate parasites.Uniting the obligate parasites seems more parsimonious thana sister group to Neodermata consisting of obligate and non-obligate parasites (Lockyer et al. 2003).

The first combined molecular analysis of parasitic andmost free-living orders of Platyhelminthes based on com-plete 18S rDNA sequences (Carranza et al. 1997) supportedthe monophyly of the Neodermata, which morphologically ischaracterized by a number of convincing synapomorphies(Ehlers 1985; Rohde 1990). Carranza et al. (1997) must alsobe credited for being the first to suggest that the sister groupto the Neodermata was not the “dalyelloid” rhabdocoels(Ehlers 1985) or the temnocephalid rhabdocoels (Brooks1989; Brooks and McLennan 1993), but a large clade of un-resolved basal and derived rhabditophorans formed byLecitoepitheliata, Prolecithophora, Proseriata, Tricladida,and Rhabdocoela (Fig. 6B). This also led to the suggestionthat parasitism in Platyhelminthes had evolved much earlierthan currently assumed. The turning point of the molecularapproach is the vast, whole-encompassing works byLittlewood et al. (1999a, 1999b) using 30 parasitic speciestogether with 52 free-living Platyhelminthes (Fig. 7D). Be-sides corroborating the monophyly and the basal position ofNeodermata within the Platyhelminthes, the works byLittlewood et al. (1999a, 1999b) provided the first reliableinternal phylogeny of the parasitic clades that showed themonophyly of Trematoda, Monogenea, and Cestoda and thatrefined the nature of the sister group to the Neodermata,which left out the Lecithoepitheliata (opposite from the re-sults of Carranza et al. 1997) but kept the Seriata. In addi-tion, Revertospermata (Joffe and Kornakova 1998) wasrejected, and a clade of ecto-/endo-parasites (Fecampiida,Urastoma, and Ichthyophaga) appeared as sister group to theTricladida and not related to Neodermata despite their simi-larities in the ultrastructure of the protonephridial flamebulb, sperm, and spermatogenesis. In summary, two main al-ternative sister groups to the Neodermata were posited: mor-phology (namely ultrastructure) suggested Fecampiida +Urastomidae as a candidate clade, whereas molecules, aswell as molecules + morphology, suggested the large cladestated above (Fig. 7D).

Littlewood and Olson (2001) expanded the database forparasitic groups up to 180 species and, together with 90rhabditophoran species and Catenulida as the outgroup,obtained the most densely sampled data set and the best

molecular-based estimate to date of the Neodermata(Fig. 7E). It strongly supported the monophyly of the Neo-dermata, Trematoda, Digenea, Cestoda, Amphilinidea,Gyrocotylidea, Monopisthocotylea, and Polyopisthocotylea,although Monogenea was not recovered as a monophyleticgroup. Likewise, it confirmed a clade of parasitic rhabdito-phoran genera (Ichthyophaga, Notentera, Urastoma, andKronborgia (INUK)), which is sister group to the Tricladida +Prolecithophora, and therefore refuted the monophyly of theRevertospermata (Kornakova and Joffe 1999; Joffe andKornakova 2001). Finally, it confirmed that the sister cladeto the Neodermata comprised the Proseriata, Rhabdocoela,INUK, Prolecithophorata, and Tricladida. That Tricladidaand Prolecithophora were sister taxa had already been sug-gested by Jondelius et al. (2001) and Baguñà et al. (2001).The latter study used 72 species of Platyhelminthes (includ-ing 5 parasitic taxa) to propose (opposite from the results ofLittlewood and Olson 2001) that the sister group to theNeodermata did not include the Proseriata, which appearedalbeit with poor support as a basal clade to them (Fig. 6E).The most recent work to date, a combined 18S + 28S rDNAanalysis (Lockyer et al. 2003), gave trees that were compati-ble with earlier studies on 18S rDNA alone (Littlewood etal. 2001b), but with stronger nodal suppport (Fig. 7F). Asin Littlewood et al. (1999b) and Baguñà et al. (2001),Proseriata was basal, monophyletic, and sister group to aclade made by the Neodermata and its sister clade formed byRhabdocoela, INUK, Prolecithophorata, and Tricladida.Finding proseriates basal to Neodermata suggests that one ofthe synapomorphy accepted for Neodermata (i.e., protone-phridial flame bulbs formed by two cells and also present inthe Proseriata) is possibly a plesiomorphy. More impor-tantly, the clade INUK did not cluster with Neodermata;hence, Revertospermata was again not supported. Finally,Monogenea was highly supported as a monophyletic clade,sister group to the Trematoda + Cestoda. The last clade, atvariance with all previously reported interrelationship of theNeodermata, was already anticipated by Mollaret et al.(1997) based on 28S rDNA sequence data. Altogether, thismeans that the Cercomeromorphae (Cestoda + Monogenea;Janicki 1920) was not supported, casting doubts on putativehomologies regarding neodermatan “cercomers” (see Chevry2002; for thorough reviews and discussions see Lockyer etal. 2003).

The origin of parasitism in PlatyhelminthesIdentifying the sister group to the Neodermata and sorting

out their interrelationships are key points to understandingthe origin and evolution of parasitism within the Platyhel-minthes. Taken at face value, the “consensus” tree of Fig. 7Fgives little insight to the condition of the proto-neodermatan.However, the strong support for a monophyletic Monogeneasister group to Trematoda + Cestoda (Lockyer et al. 2003;Fig. 7E) indicates ectoparasitism (as seen in monogeneans)as the plesiomorphic condition, endoparasitism appearinglater on within the lineage that gave rise to extant trematodesand cestodes. This molecular monophyly (despite all previ-ous 18S rDNA results resolving the Monogenea as para-phyletic; see thorough discussions in Littlewood et al. 2001band Lockyer et al. 2003) agree with the monophyly ofmonogeneans based on several morphological synapomor-

© 2004 NRC Canada

182 Can. J. Zool. Vol. 82, 2004

phies: two pair of eyes, three bands of ciliary patches, onepair of ventral anchors, and one egg filament (Boeger andKritsky 2001). In addition, monogeneans are structurallysimilar to rhabdocoels, some bearing a protrusible pharynxand having extracorporeal and intracellular digestion.

Neodermatans are characterized by several synapomor-phies: replacement of the larval epidermis by a neodermis,lack of vertical ciliary rootlets of epidermal cilia, presenceof specific electrodense collars of sensory receptors,axonemes of sperm incorporated into the sperm body, andincorporation of vertebrate host in life cycle either as a sin-gle host (Monogenea), as a facultative host (some Aspido-gastrea), or usually as an obligate final host (all the others)(Littlewood et al. 1999b). The neodermis is its most peculiarstructural feature. According to Tyler and Tyler (1997), theneodermis has to met four structural criteria: (1) syncytyal,(2) unciliated, (3) insunk (i.e., its nuclei lie below the base-ment membrane), and (4) having multiple branching connec-tions between the epidermal perikarya and the surface layer.Among free-living rhabditophorans, namely within the sistergroup to the Neodermata (Fig. 7F), parasitism and othersymbiotic permanent associations with invertebrate hostshave evolved repeatedly (Cannon 1998). With the exceptionof the INUK clade, most species retain a ciliated epidermisand other morphological and physiological features of thefree-living Rhabditophora. Instead, species of the INUKclade bear some structural and physiological (e.g., absorp-tion of food through the epidermis, shift to a glycogen stor-age, high fecundity) features of neodermatans, but they stilllack a true neodermis. However, molecular data reject theINUK clade as sister group to the Neodermata. Finally, theprolecithophoran Genostoma spp. (a symbiont of crusta-ceans) has met the four criteria, but it is not a member of theNeodermata because it does not share the other neodermatansynapomorphies (Tyler and Tyler 1997) while keeping mostof the features of free-living rhabditophorans.

Altogether, this indicates that within the sister group tothe Neodermata (formed by rhabdocoels, prolecitophorans,triclads, and the INUK clade) some features may ease the re-peated, though infrequent (just 200 species compared withthe 50 000 or so neodermatans; for an interesting explana-tion see Rohde 1997), adoption of a parasitic life style.Because these groups are not direct sister groups to Neo-dermata, it is important to understand the likelihood of con-vergence. In other words, are there many developmentalways to become a parasite or does its evolution depend on aprescribed, limited set of mutational events without whichthe opportunity for the evolution of parasitism is not avail-able? Answering this question, together with finer phylogen-etic analyses, would allow one to know which features(genetic or not) made Neodermata monophyletic to the ex-clusion of other obligate parasitic clades. In addition, it mayhelp to test Rohde’s hypothesis (1997) of whether the infre-quency of adoption of the parasitic life style by rhabdito-phorans other than Neodermata is due to being actually“trapped” in a very specific adaptational peak or whether itis due to neodermatan competition.

In the meantime, and awaiting comparative full-scalegenomic sequencing and functional analyses between closelyrelated free-living and parasitic Platyhelminthes, the mainapproach has been to look for preadaptational features in

free-living taxa. Tyler and Tyler (1997) have proposed thatepidermal replacement during embryogenesis in many tur-bellarians may be a preadaptation used by the parasites inthe formation of the neodermis. However, evidence for thisassumption is scanty. Moreover, epidermal replacementin all Platyhelminthes is a continuous process occurringthroughout life (Ladurner et al. 2000) and has a clearadaptative role in withstanding long periods of starvationwith degrowth followed by growth periods leading to the ex-pansion of the epidermis (and other tissues and organs) bymigrating cells (neoblasts; Baguñà 1981) from the paren-chyma. An additional feature worth exploring is the parallel-ism between the neodermis and the epithelia from thepharynx cavity and the outer and inner pharyngeal epitheliaof freshwater triclads (Baguñà 1973; Bowen and Ryder 1973)and of kalyptorhynchian rhabdocoels (De Vocht 1989). Thefirst two epithelia, of ectodermal origin, are insunk, sparselyciliated, and microvillus and glycogen rich, albeit cellular.At the distal part of the pharynx, the outer epithelium givesway to the inner epithelium which is insunk, unciliated, verymicrovillus rich, and with an extremly infolded basal partpacked with glycogen granules. As with the neodermis, pha-ryngeal epithelia with insunk perikarya may protect the sur-face layer during feeding from abrasions and the action ofenzymes and prey defenses. Finally, an even more promis-ing case to explore is the epidermis replacement inproseriates. Their adult epidermis is insunk and arises fromneoblasts that send processes to the surface to produce a cili-ated surface layer connected to insunk perykaria (Giesa1966). The position of proseriates basal to Neodermata fos-ter the exploration of such a connection. Regarding the evo-lutionary mechanisms involved, it is tempting to suggest thatgenes or gene networks at the base of such “preadaptations”may have been co-opted by epidermal cells during the evo-lution of neodermatans from proto-neodermatan looking likeextant proseriates.

Did the first neodermatan adopt an ectoparasitic way oflife or was it an endoparasite from the very beginning? Be-cause all Neodermata have in common a vertebrate host andbecause endoparasitism is considered the plesiomorphicstate, a common ancestor endoparasite of vertebrates has un-critically been taken for granted (Brooks 1989; Brooks andMcLennan 1993; Littlewood et al. 1999b, 2001b). However,the new position of a monophyletic Monogenea as sistergroup to Trematoda + Cestoda suggests an alternative sce-nario with ectoparasitism as the plesiomorphic condition andendoparasitism appearing just once with the divergence ofthe Trematoda and Cestoda. However, an endoparasitic an-cestor that shifted to ectoparasitism in the lineage giving riseto the monogeans could not be ruled out, as it requires thesame number of steps (for discussions on former models seeLittlewood et al. 2001b and Rohde 2001). Given the earlyappearance of parasitism in Platyhelminthes and the longtime elapsed since it occurred, knowledge of the first hostsfrom those extant parasites is anyone’s guess. As pointed outlong ago by Llevellyn (1965), the earliest parasite may haveadapted to feed on the skin of several bottom-dwelling slug-gish invertebrates, with vertebrates being incorporated in thelife cycle later. Development of posterior adhesive pads orhaptors (i.e., there are striking convergences in advancedtriclads such as dendrocoelid freshwater triclads and land

© 2004 NRC Canada

Baguñà and Riutort 183

planarians) and later on marginal hooks, as temporary orpermanent attachments, may have enhanced feeding oppor-tunities and may have led to internal epithelia to endoparasitism.

The internal phylogeny of selected rhabditophoranorders

In the last 10 years, molecular analyses that have beenbased namely on 18S rDNA sequence data have been ap-plied to several rhabditophoran orders. A detailed account ofall of them is out of the scope of this review; hence, only themost interesting and thorough studies are considered.

Outside the Rhabditophora, only the Acoela has been ana-lysed in detail (Table 2). An extensive internal phylogenyfrom 18S rDNA sequence data appeared in Jondelius et al.(2002). However, Hooge et al. (2002) was the first studydevoted to the molecular phylogeny of the group using com-plete sequences from 32 acoel species. Interestingly, Parato-mellidae and Solenofilomophidae grouped basally to theother acoel species, which was in agreement with Ehlers’s(1992) proposal of the Paratomellidae as sister group to therest of acoels. Moreover, several large families, namelyConvolutidae and Haploposthiidae, appeared polyphyleticallyall over the phylum, whereas Childiidae and Otocelididaehad unstable positions that call for deep reasessments of tra-ditional, morphologically based phylogenies. The 18S rDNAtree had some interesting paralellisms with trees providedfrom sperm ultrastructure (Raikova et al. 2001) and with pat-terns of body musculature (Hooge et al. 2002) (Fig. 8). Inany case, more data, including sequences from the 28SrDNA and other genes, are needed.

Within the Rhabditophora, Polycladida has been barelystudied (only partial 28S rDNA sequences reported inLitvaitis and Newman 2001), and no data is available fromMacrostomorpha (Macrostomida + Haplopharyngida) andLecithoepitheliata. Regarding the order Seriata (Proseriata +Tricladida; Ehlers 1985), its monophyly has been questionedboth on morphological and molecular grounds (Sluys 1989;Carranza et al. 1997; Littlewood et al. 1999a) and there isample consensus that they form two unrelated clades (seeFigs. 6B–6F). Within Proseriates (Curini-Galletti 2001) (Ta-ble 3), both its monophyly as well as their outgroup relation-ships are uncertain. Ingroup relationships have beenanalyzed based on the first variable domains of 28S rDNA(D1–D3) and a family-level morphological matrix, undermaximum-parsimony and minimum-evolution analyses(Littlewood et al. 2000). An analysis of the family Mono-celiidae based on the domains D3–D6 have been providedby Litvaitis et al. (1996). The first study placed a clade con-sisting of Unguiphora + Coelogynoporidae external to agroup that included all the other lithophorans. The support inthe minimum-evolution tree, however, was very poor, andmorphology-based and molecular trees differed substantially,namely in the position of the Coelogynoporidae. Therefore,molecular analyses of Proseriates need wider sampling to in-corporate 18S rDNA and other gene-sequence data. Interest-ingly, a former proseriate, Bothrioplana semperi, consideredby Sopott-Ehlers (1985) as sister group to the Tricladida felloutside the Tricladida and the Proseriata but was basal to theProseriata, although with low support (Baguñà et al. 2001;see Fig. 6E). Despite being one of the most speciose orders,Rhabdocoela is still poorly known from a molecular point of

view. Partial phylogenies have been included phylum-wideanalyses (Littlewood et al. 1999a, 1999b; Zamparo et al.2001). Among the big suprafamilial clades, Kalyptorhynchiaappears basal in most analyses, Dalyellida and Temno-cephalida are monophyletic, whereas the Typhloplanida ispolyphyletic.

The consensus tree of the Platyhelminthes (Fig. 6F) sup-ports the Prolecithophora and Tricladida as sister groups andthe most derived rhabditophorans. Prolecithophorans havebeen amply studied by Jondelius et al. (2001) and Norén andJondelius (2002), using complete 18S rDNA and partial 28SrDNA sequences from 25 species. The agrupations Com-binata and Separata (Karling 1940) were not retrieved. In-stead, Plagiostomidae was most basal, sister group to a newclade called Pseudostomiidae formed by different families.Further studies are needed. With the exception of theNeodermata, the Tricladida (Table 3) has been the moststudied rhabditophoran order. Overviews of the group havebeen provided by Carranza et al. (1998a) and Baguñà et al.(2001) showing Tricladida as a monophyletic taxon not re-lated to the Proseriata; therefore, the validity of the Seriata isrejected. In addition, the later study, which used neighbor-joining and maximum-likelihood trees, showed a sister-grouprelationship between the Tricladida and the Prolecithophora;both of them forming a sister clade to the INUK clade (seeFig. 6E–6F and Fig. 9). Moreover, taking advantage of asynapomorphic event involving the 18S ribosomal gene(Carranza et al. 1996) together with the complete 18S rDNAand some mitochondrial genes of up to 30 species, it wasshown that the infraorder Maricola (marine triclads) is the

© 2004 NRC Canada

184 Can. J. Zool. Vol. 82, 2004

Acoelomorpha Ehlers, 1985Acoela Uljanin, 1870

Paratomella Ehlers, 1992Family Paratomellidae

Euacoela Ehlers, 1992Family Actinoposthiidae

AnaperidaeAntigonaridaeAntroposthidaeChildiidaeConvolutidaeDiopisthoporidaeHallangidaeHaploposthiidaeHofsteniidaeMecynostomidaeMyostomellidaeNadinidaeOtocelididaeProporidaeSagittiferidaeSolenofilomorphidaeTaurididae

Nemertodermatida Karling, 1940Family Ascopariidae Sterrer, 1998

Nemertodermatidae Steinböck, 1930

Note: Current classification is based on Ehlers (1985, 1992), Tyler andBush (2001), and Hooge et al. (2002).

Table 2. Current classification of the phylum Acoelomorpha.

most basal, whereas the Paludicola (freshwater triclads) isparaphyletic, with the family Dugesiidae being a sister groupto the Terricola (land planarians) to the exclusion of theother two paludicolan families Planariidae and Dendro-coelidae (Carranza et al. 1998a) (Table 3 and Fig. 9). A newproposed clade, the Continenticola, groups the present fami-lies Dugesiidae, Planariidae, and Dendrocoelidae with theland planarians (the Terricola). The monophyly of theTerricola and the Dugesiidae, however, could not be vali-

dated (Carranza et al. 1998b). Finally, the internal phylo-genies of the Dugesiidae and the Terricola are at present be-ing pursued through a multigenic approach using 18S + 28S+ mitochondrial gene sequences.

Of the 350 complete 18S rDNA sequences, over 270 referto parasitic Platyhelminthes or Neodermata (Lockyer et al.2003). Besides, complete or long stretches of the 28S geneas well as the EF1-alpha are also available. However, thevastness of the problems encountered, the conflicts pro-

© 2004 NRC Canada

Baguñà and Riutort 185

Fig. 8. 18S rDNA tree of the interrelationships of the Acoela onto which two morphological characters (sperm morphology and pat-terns of body-wall musculature) have been superimposed (modified in a simplified form from Hooge et al. 2002, which incorporatedata from Raikova et al. 2001). For current classification of the Acoela see Table 2. Structure of the spermatozoa is indicated by dot-ted rectangles. Character state changes of body-wall musculature and sperm structure are indicated by small vertical solid black rectan-gles. 1, U-shaped muscles and longitudinal muscles that wrap around posterior rim of mouth; 2, dorsal cross-over muscles and ventralcross-over muscles; and 3, change from cortical to axial microtubules. Note that several families (e.g., Convolutidae andHaploposthiidae) are polyphyletic, whereas others are in need of revision. For a detailed account of both sets of characters see Raikovaet al. (2001) and Hooge et al. (2002).

duced, and the hypotheses suggested regarding the internalrelationships of the group are out of the scope of this review.Readers are referred to the excellent monograph edited byLittlewood and Bray (2001), as well as to the paper byLockyer et al. (2003) that offers a new and fresh view of theNeodermata’s internal phylogeny.

Methodological caveats

The danger of monogenic phylogenies and the need ofa multigenic approach

18S rDNA has proven to be a very good phylogeneticmarker. Witnesses are the answers given to old taxonomicand phylogenetic problems where morphology and embryol-ogy failed. Even so, it is not a trouble-free molecule. Firstly,and despite providing a new phyogenetic framework for thewhole Metazoa (Adoutte et al. 1999, 2000), it has been un-

able to sort out, among others, the order among the three bigsuperclades Ecdysozoa, Lophotrochozoa, and Deuterostomiaand the internal relationships within the Lophotrochozoa(see Fig. 3B). Secondly, as is often pointed out (Baldauf andPalmer 1993; Phillipe et at. 1994; Abouheif et al. 1998), thephylogenetic information contained within the 18S rDNAmolecule falls short of dividing the major groups or erectingnew phyla. These problems have recently been tackled andpartially solved using new and more sophisticated evolutionarymodels of phylogenetic inference. In particular, maximum-likelihood methods together with increasing the number ofparameters analysed (gamma distribution, covarion models,etc.) have made molecular trees based on 18S rDNA morereliable in the last few years.

Nevertheless, some uncertainties still remain that call fornew corroborative data. A major question is whether increas-ing the length of the sequence or increasing the number ofsampled taxa is the best approach to overcome the lack ofsupport (and hence lack of resolution) for certain taxonomicgroups and for LBA artifacts. Addition of more species, es-pecially those selected to break long branches, appear to bea better approach under simulation than mere addition ofnew gene sequences (Graybeal 1998; Zwickl and Hillis2002). However, real data were extremely variable. For 18S-rDNA-based trees (the evolution in the position of Platy-helminthes within the metazoan being a good example), add-ing further species increased its resolution and robustness.However, this correlation seems to have an upper boundarybeyond which noise overcomes the information added, aswell as increasing the computational effort and complexity.Instead, adding new sequences increase resolution and reli-ability (Baldauf et al. 2000; Regier and Shultz 2001;Bapteste et al. 2002; Ruiz-Trillo et al. 2002), with the addi-tional bonus of providing data that are independent of the18S rDNA to test the results obtained.

However, protein-coding genes are less universal, moredifficult to amplify by polymerase chain reactions, and oftenshorter and less information-rich than rRNA genes. More-over, trees from 18S rDNA sequences and those inferredfrom individual protein-coding genes (or from their proteins)are often different and impossible to match. Such differencesmay result from (i) noise and bias obscuring the phylogen-etic signal in protein-based trees; (ii) wide disparities in evo-lutionary rates among lineages; (iii) different phylogenetichistories among genes (e.g., lateral gene flow or gene dupli-cation followed by random independent gene loss); and(iv) uneven taxonomic sampling and (or) inadequate charac-terization of currently available protein data sets. As it hap-pened 10 years ago for 18S rDNA trees, some of thesehurdles will be overcome by increasing the sampling effort,using better evolutionary models, and namely, by adopting amultigenic strategy to sequence nuclear genes other than 18Sand 28S rDNA.

Recent results from multiple-gene analysis (Baldauf et al.2000; Regier and Shultz 2001; Bapteste et al. 2002; Ruiz-Trillo et al. 2002) show the advantages of this approach overpiling up sequences of a single gene from additional species.This conclusion stems from realizing that althoughbootstrap-proportion values for particular groups varywidely across genes in ways not reflecting the total numberof characters, a multigenic approach generally improves

© 2004 NRC Canada

186 Can. J. Zool. Vol. 82, 2004

(a) Morphology.

Order Seriata Bresslau, 1933Suborder Proseriata Meixner, 1938

Infraorder Unguiphora Sopott-Ehlers, 1985Family Nematoplanidae

Infraorder Lithophora Steinböck, 1925Family Archimonocelididae

CoelogynoporidaeMonocelididaeMotoplanidaeOtomesostomidaeOtoplanidae

Suborder Bothrioplanida Sopott-Ehlers, 1985Tricladida Lang, 1884

Infraorder Maricola Hallez, 1892Cavernicola Sluys, 1990Terricola* Hallez, 1892Paludicola Hallez, 1892

Family Dugesiidae* Ball, 1974Planariidae Stimpson, 1857Dendrocoelidae Hallez, 1894

(b) Molecules (18S rDNA + Cox I).b

Order TricladidaInfraorder Maricola Hallez, 1892

Cavernicola Sluys, 1990“Continenticola” nom. nud. (Carranza at al. 1998a)

Family Dugesiidae* nom. nud.Infraorder “Terricola”* nom. nud.

Family Planariidae nom. nud.Dendrocoelidae nom. nud.

aBased on Curini-Galletti (2001) and Baguñà et al. (2001).bBased on sequences of the ribosomal gene 18S rDNA and the mito-

chondrial gene cytochrome oxidase I (Cox I) (Carranza et al. 1998a;Baguñà et al. 2001). Proseriata and Bothrioplanida did not cluster in mo-lecular trees with the Tricladida. Hence, the order Seriata is rejected.

*The infraorder Terricola and the family Dugesiidae, using moleculardata to cluster, formed a sister group to the families Planariidae andDendrocoelidae. Therefore, Paludicola is paraphyletic and not a validtaxon being replaced by the new infraorder Continenticola (nom. nud.),which comprises the former Paludicola and Terricola (see Fig. 9).

Table 3. Current classification of the order Seriata, suborderProseriata, and suborder Tricladida.a

© 2004 NRC Canada

Baguñà and Riutort 187

Fig. 9. Contrasting phylogenetic hypothesis for the order Tricladida based on molecular data (Carranza et al. 1998a; Baguñà et al.2001) (a) and based on morphological characters (adapted from Sluys 1989) (b). For current classification of the order Tricladida seeTable 3. The molecular tree in Fig. 9a illustrates the monophyly of the order Tricladida and its sister-group relationships to the orderProlecithophora, the monophyly of the infraorder Maricola, and the recently proposed clade, the infraorder Continenticola, grouping thepresent families Dugesiidae, Planariidae, Dendrocoelidae, and the infraorder Terricola (land planarians). For the sake of clarity theinfraorder Cavernmicola (Sluys 1990) is not included. Note the clade formed by the family Dugesiidae and the infraorder Terricola,based on characters 14 and 15. Selected morphological characters from Sluys (1989) and the molecular apomorphy (character 15) havebeen mapped onto the tree. The morphologically based tree depicted in Fig. 9b illustrates the internal phylogeny of the suborderTricladida, sister group to the suborder Proseriata, within the order Seriata (from Ehlers 1985). For the sake of clarity the suborderBothrioplanida (Sopott-Ehlers, 1985) and the infraorder Cavernmicola (Sluys 1990) are not included. Characters (a, b, c) defining theinfraorder Paludicola, which appears paraphyletic in the molecular tree, are also depicted. Character d (dugesiid eye structure), anapomorphy of the family Dugesiidae, is now considered a synapomorphy for the Dugesiidae–Terricola clade (character 14 in Fig. 9a).1, tricladoid intestine; 2, crossing-over of pharynx muscles; 3, embryology; 4, cerebral position of female gonads; 5, serial arrangementof many nephridiopores; 6, marginal adhesive zone; 7, Haftpapillen in annular zone; 8, loss of Haftpapillen; 9, resorptive vesicles; 10,reduction in number of longitudinal nerve cords; 11, common oviduct opening into atrium; 12, dendrocoelid pharyngeal musculature;13, anterior adhesive organ; 14, multicellular eye cup with numerous retinal cells; 15, two types of 18S rDNA genes (type I and typeII); 16, creeping sole; 17, diploneuran nervous system; a, four subepidermal muscle layers; b, spermatophore; c, probursal condition;and d, dugesiid eye (= character 14). Dotted lines indicate groups that are not well supported in the molecular phylogenetyic analysis.

node support relative to single-gene analyses. In otherwords, each gene bears as a phylogenetic marker a uniqueset of strengths and weaknesses; therefore, it is unlikely thateach gene by itself will ever be able to strongly, or perhapseven accurately, resolve all deep branches of a universaltree. The multigenic approach is thus particularly usefulwhen the main goal is to resolve a large number of nodes atdeep taxonomic levels. However, it is also important to warnagainst a too strict adherence to a multigenic approach. Eachset of data should be analysed carefully and positions show-ing saturation or other kinds of noise clearly avoided (e.g.,third positions of codons in certain protein coding genes). Itwill also be important to develop methods and evolutionarymodels that pay more attention to problems derived from thesimultaneous use of different genes with different evolution-ary features.

The search for molecular synapomorphiesAnother source of data to test the 18S rDNA results are

the emerging set of molecular characters that originate fromlarge genome or gene rearrangements. These differ from,and have the advantage over, primary sequence data of beingqualitative characters that correspond to rare or uniqueevents which are likely not homoplasic (Rokas and Holland2000). Such characters include rearrangements of mitochon-drial gene order (Boore and Brown 1998; but see Le et al.2000), gene duplications and divergence within broadly con-served chromosomal arrays or gene families such as the Hoxcluster (Garcia-Fernandez and Holland 1994; Falciani et al.1996; Holland and Garcia-Fernandez 1996; Telford 2000),transposition of mobile elements inside the genome (Hillis1999; Miyamoto 1999; Nikaido et al. 1999), and the modi-fied codon usage in mitochondrial genome (Telford et al.2000). On a finer phylogenetic scale, some qualitative mark-ers have proven invaluable. Within the Platyhelminthes, wecould mention, among others, the satellite repeats that arespecific for species of the Dugesia gonocephala s.l. speciescomplex (Batistoni et al. 1998), the presence of two types of18S rDNA in the Dugesiidae and the Terricola (orderTricladida) (Carranza et al. 1996), and specific indels withinthe ITS-1 gene of the ribosomal complex that are species-specific for species of the genus Dugesia (Dugesiidae; orderTricladida) and that are very useful in assigning asexualforms to its sexual counterparts (Baguñà et al. 1999). Furtherexamples are the large insertions in segments E23 and E43in 18S rDNA in all Neodermata (Littlewood et al. 1999a), aswell as numerous examples of indels in different species be-longing to the Tricladida, Prolecithophora, and Rhabdocoela(Joffe and Kornakova 2001).

Are total-evidence analyses the panacea?If one feels trapped within this molecular turmoil, it is ex-

tremely important not to overlook morphological, embryo-logical, and other data that until very recently were the stuffof all phylogenetic trees. The “big” question posed is “howshould molecules and morphology be handled together toget more reliable phylogenies?” As applied to Platyhelmin-thes, neither total-evidence analyses (Littlewood et al.1999a) nor “matrix representation with parsimony” super-trees (Wilkinson et al. 2001) are the panacea to deal withsuch heterogeneous sets of data. However, whereas methods

handling complex evolutionary models (e.g., maximum-likelihood and Bayesian inference analyses) are best suitedto extract information from gene data sets, they are not sosuited to deal with molecular and classical synapomorphieswhere parsimony or completely different models of evolu-tion are more at ease. Therefore, a good compromise wouldbe to take into account all the data to first construct a molec-ular tree to which, at a later date, molecular and classicalsynapomorphies are mapped. The latter can help to resolveuncertain nodes (by the existence of a synapormophy) or de-tect inconsistencies in the molecular tree (groupings that areincompatible with a clear synapomorphy).

Systematic revision

From the molecular (and morphological) evidence broughtforward in the last decade it seems increasingly likely thatwhile many of the Platyhelminthes (Catenulida + Rhab-ditophora) belong to the superclade Lophotrochozoa, theAcoelomorpha (Acoela + Nemertodermatida) are genuinelybasal bilaterians. Hence, present day Platyhelminthes (seeTable 1) are likely to be polyphyletic (see Fig. 5). Therefore,the Acoelomorpha merits to be recognized as a new phylum,whereas the name Platyhelminthes should be adscribed topresent Catenulida and Rhabditophora, which despite lack-ing clear morphological synapomorphies, seem to form amonophyletic group.

Phylum Acoelomorpha, new phylum (synonym: Acoelo-morpha Ehlers, 1985)

DIAGNOSIS. Acoelomate; unsegmented marine metazoanswith bilateral symmetry; three embryonic body layers; blind-ending gut with no permanent cavity (acoel); subepithelialnervous system concentrated at the anterior body end form-ing a primitive brain; and no respiratory, circulatory, andexcretory systems. Main autapomorphies are the intercon-necting rootlets of epidermal cells that form a distinctivenetwork, epidermal cilia that bear a shaft region, and embry-onic cleavage of duet spiral type. The extracellular matrix ishigly reduced and lack protonephridia (likely a plesiomor-phic condition). Hermaphrodites are without distinctivegonads and reproduction is sexual and asexual. Direct devel-opment with no known larval stages. Free-living or symbi-otic, marine.

ETYMOLOGY. Acoel meaning without a coel or true alimen-tary canal; coel from the Greek for “cavity” and morph fromthe Greek for “form”.

TAXONOMICAL REMARKS. Comprises two former orders of thephylum Platyhelminthes (sensu Ehlers 1985): the AcoelaUljanin, 1870 and the Nemertodermatida Karling, 1940 (seeTables 1 and 2, Fig. 2)

Phylum Platyhelminthes (synonym: Platyhelminthes Minot,1876)

DIAGNOSIS. Acoelomate; unsegmented marine metazoanswith bilateral symmetry; three embryonic body layers; blind-ending gut with definite cavity; and a nervous system with adistinctive brain with neuropile and asymmetrically distrib-uted (namely ventral) longitudinal nerve cords. With pro-

© 2004 NRC Canada

188 Can. J. Zool. Vol. 82, 2004

tonephridia, but it has no respiratory and circulatoorysystems. Main autapomorphy is a rostral–caudal/verticalrootlet system of epidermal cells without accessory centrioles.They are mostly hermaphroditic with well-developed gonadsand sperm that are usually biflagellate. Development is usu-ally direct with cleavage of quartet spiral type except insome families of polyclads that bear different sorts of larvaeand in parasitic species where numerous secondary larvalstages may occur. Asexual multiplication is widespread.Free-living, symbiotic, or parasites from marine, freshwater,and terrestrial habitats.

ETYMOLOGY. Platy is Greek for “flat” and heminthes is Greekfor “worms”.

TAXONOMIC REMARKS. Comprises two main clades: Catenulidavon Graff, 1905 and Rhabditophora Ehlers, 1985 (see Ta-ble 1, Fig. 2).

Acknowledgements

We are very grateful to Iñaki Ruiz-Trillo and Jordi Papsfor their invaluable help with illustrations and for thoughtfuldiscussions. This work was funded by grants from theGeneralitat de Catalunya, Nos. 1999SGR-00026 and2001SGR-00102 (to J.B).

References

Abouheif, E., Zardoya, R., and Meyer, A. 1998. Limitations ofmetazoan 18S rRNA sequence data: implications for recon-structing a phylogeny of the animal kingdom and inferring thereality of the Cambrian explosion. J. Mol. Evol. 47: 394–405.

Adoutte, A., Balavoine, G., Lartillot, N., and de Rosa, R. 1999.Animal evolution: the end of the intermediate taxa? TrendsGenet. 15: 104–108.

Adoutte, A., Balavoine, G., Lartillot, N., Lespinet, O.,Prud’homme, B., and de Rosa, R. 2000. The new animal phy-logeny: reliability and implications. Proc. Natl. Acad. Sci.U.S.A. 97: 4453–4456.

Aguinaldo, A.M.A., Turbeville, J.M., Lindford, L.S., Rivera, M.C.,Garey, J.R., Raff, R.A., and Lake, J.A. 1997. Evidence for aclade of nematodes, arthropods and other moulting animals.Nature (Lond.), 387: 489–493.

Ahlrichs, W.H. 1995. Seison annulatus and Seison nebaliae —Ultrastruktur und Phylogenie. Verh. Dtsch. Zool. Ges. 88: 155.

Ax, P. 1985. The position of Gnathostomulida and Platyhelminthesin the phylogenetic system of the Bilateria. In The origins andrelationships of lower invertebrates. Edited by S. Conway Mor-ris, J.D. George, R.Gibson, and H.M. Platt. Oxford UniversityPress, Oxford. pp. 168–180.

Ax, P. 1987. The phylogenetic system. John Wiley & Sons,Chichester, UK.

Ax, P. 1996. Multicellular animals. A new approach to the phylo-genetic order in nature. Vol. I. Springer-Verlag, Berlin, Ger-many.

Ax, P., and Dörjes, J. 1966. Oligochoerus limnophilus nov.spec.,ein kaspisches Faunenelement als erster Süßwasser-vertreter derTurbellaria Acoela in Flüssen Mitteleuropas. Int. Rev. Ges.Hydrobiol. 57: 15–44.

Baguñà, J. 1973. Estudios citotaxonómicos, ecológicos e histofisio-logía de la regulación morfogenética durante el crecimiento y laregeneración de la raza asexuada de la planaria Dugesia

mediterranea n.sp. (Turbellaria: Tricladida: Paludicola). Ph.D.thesis, University of Barcelona, Barcelona.

Baguñà, J. 1981. Planarian neoblast. Nature (Lond.), 290: 14–15.Baguñà, J., Saló, E., and Auladell, C. 1989. Regeneration and pat-

tern formation in planarians. III. Evidence that neoblasts aretotipotent stem-cells and the source of blastema cells. Develop-ment (Camb.), 107: 77–86.

Baguñà, J., Carranza, S., Pala, M., Arnedo, M.A., Giribet, G.,Ribera, C., Riutort, M., and Ribas, M. 1999. From morphologyand karyology to molecules. New methods for taxonomic identi-fication of asexual populations of freshwater planarians. A trib-ute to Professor Mario Benazzi. Ital. J. Zool. 66: 207–214.

Baguñà, J., Carranza, S., Paps, J., Ruiz-Trillo, I., and Riutort, M.2001. Molecular taxonomy and phylogeny of Tricladida. In Theinterrelationships of the Platyhelminthes. Edited by D.T.J.Littlewood and R.D. Bray. Taylor & Francis, London, England.pp. 49–56.

Balavoine, G. 1997. The early emergence of platyhelminths is con-tradicted by the agreement between 18S rRNA and Hox genesdata. C.R. Acad. Sci. Ser. III Sci. Vie, 320: 83–94.

Balavoine, G. 1998. Are Platyhelminthes coelomates without acoelom? An argument based on the evolution of Hox genes.Am. Zool. 38: 843–858.

Baldauf, S.L., and Palmer, J.D. 1993. Animals and fungi are eachother’s closest relatives: congruent evidence from multiple pro-teins. Proc. Natl. Acad. Sci. U.S.A. 90: 11 558 – 11 562.

Baldauf, S.L., Roger, A.J., Wenk-Siefert, I., and Doolittle, W.F.2000. A kingdom-level phylogeny of eukaryotes based on com-bined protein data. Science (Wash., D.C.), 290: 972–977.

Bapteste, E., Brinkmann, H., Lee, J.A., Moore, D.V., Sensen, C.W.,Gordon, P., Durufle, L., Gaasterland, T., Lopez, P., Muller, M.,and Philippe, H. 2002. The analysis of 100 genes supports thegrouping of three highly divergent amoebae: Dictyostelium,Entamoeba, and Mastigamoeba. Proc. Natl. Acad. Sci. U.S.A.99: 1414–1419.

Batistoni, R., Filippi, C., Salvetti, A., Cardelli, M., and Deri, P.1998. Repeated DNA elements in planarians of the Dugesiagonocephala group (Platyhelminthes, Tricladida). Hydrobiologia,383: 139–146.

Bayascas, J.R., Castillo, E., and Saló, E. 1998. Platyhelmintheshave a Hox code diferentially activated during regeneration, withgenes closely related to those of spiralian protostomes. Dev.Genes Evol. 208: 467–473.

Berney, C., Pawlowski, J., and Zaninetti, L. 2000. Elongation Fac-tor 1 – alpha sequences do not support an early divergence ofthe Acoela. Mol. Biol. Evol. 17: 1032–1039.

Boeger, W.A., and Kritsky, D.C. 2001. Phylogeny of the Mono-genoidea: a critical review of hypothesis. In The interrelation-ships of the Platyhelminthes. Edited by D.T.J. Littlewood andR.D. Bray. Taylor & Francis, London, England. pp. 92–102.

Boore, J.L., and Brown, W.M. 1998. Big trees from little genomes:mitochondrial gene order as a phylogenetic tool. Curr. Opin.Genet. Dev. 8: 668–674.

Bowen, I.D., and Ryder, T.A. 1973. The fine structure of theplanarian Polycelis tenuis Iijima. I. The pharynx. Protoplasma,78: 223–241.

Boyer, B.C., Henry, J.Q., and Martindale, M.Q. 1996. Dual originsof mesoderm in a basal spiralian: cell lineage analyses in thepolyclad turbellarian Hoploplana inquilina. Dev. Biol. 179:329–338.

Boyer, B.C., Henry, J.J., and Martindale, M.Q. 1998. The cell lin-eage of a polyclad turbellarian embryo reveals close similarityto coelomate spiralians. Dev. Biol. 204: 111–123.

© 2004 NRC Canada

Baguñà and Riutort 189

Brooks, D.R. 1989. A summary of the database pertaining to thephylogeny of the major groups of parasitic platyhelminthes,with a revised classification. Can. J. Zool. 67: 714–720.

Brooks, D.R., and McLennan, D.A. 1993. Macroevolutionary pat-terns of morphological diversification among parasitic flat-worms (Platyhelminthes, Cercomeria). Evolution, 47: 495–509.

Brusca, R.C., and Brusca, G.J. 1990. Invertebrates. Sinauer Associ-ates Inc., Sunderland, Mass.

Campos, A. Cummings, MP., Reyes, J.L., and Laclette, J.P. 1998.Phylogenetic relationships of Platyhelminthes based on 18S ri-bosomal gene sequences. Mol. Phylogenet. Evol. 10: 1–10.

Cannon, L.R.G. 1998. Turbellaria of the world. A guide to familiesand genera. Version 1.0 [CD-ROM]. Amsterdam: Expert Centerfor Taxonomic Identification, University of Amsterdam, Amster-dam.

Carranza, S., Giribet, G., Ribera, C., Baguñà, J., and Riutort, M.1996. Evidence that two types of 18S rDNA coexist in the ge-nome of Dugesia (Schmidtea) mediterrranea (Platyhelminthes,Turbellaria, Tricladida). Mol. Biol. Evol. 13: 824–832.

Carranza, S., Baguna, J., and Riutort, M. 1997. Are the Platy-helminthes a monophyletic primitive group? An assessment us-ing 18S rDNA sequences. Mol. Biol. Evol. 14: 485–497.

Carranza, S., Littlewood, D.T.J., Clough, K.A., Ruiz-Trillo, I.,Baguñà, J., and Riutort, M. 1998a. A robust molecular phylog-eny of the Tricladida (Platyhelminthes: Seriata) with a discus-sion on morphological synapomorphies. Proc. R. Soc. Lond. BBiol. Sci. 265: 631–640.

Carranza, S., Ruiz-Trillo, I., Littlewood, D.T.J., Riutort, M., andBaguñà, J. 1998b. A reappraisal of the phylogenetic and taxo-nomic position of land planarians (Platyhelminthes, Turbellaria,Tricladida) inferred from 18S rDNA sequences. Pedobiologia,42: 433–440.

Cavalier-Smith, T. 1998. A revised six-kingdom system of life.Biol. Rev. (Camb.), 73: 203–266.

Chevry, L. 2002. The terminology of larval cestodes or metacesto-des. Syst. Parasitol. 52: 1–33.

Cook, C.E., Jiménez, E., Akam, M., and Saló, E. 2004. The Hoxgene complement of Acoel flatworms, a basal bilaterian clade.Evol. Dev. In press.

Curini-Galletti, M. 2001. The Proseriata. In The interrelationshipsof the Platyhelminthes. Edited by D.T.J. Littlewood and R.D.Bray. Taylor & Francis, London, England. pp. 41–48.

De Robertis, E.M. 1997. The ancestry of segmentation. Nature(Lond.), 387: 25–26.

de Rosa, R., Grenier, J.K., Andreeva, T., Cook, C., Adoutte, A.,Akam, M., Carroll, S.B., and Balavoine, G. 1999. Hox genes inbrachiopods and priapulids: implications for protostome evolu-tion. Nature (Lond.), 399: 772–776.

De Vocht, A.J-P. 1989. Ultrastructure of the proboscis in Cysti-planidae, (Platyhelminthes, Kalyptorhynchia). Zoomorphology(Berl.), 109: 1–10.

Dewel, R.A. 2000. Colonial origin for eumetazoa: major morpho-logical transitions and the origin of bilaterian complexity. J.Morphol. 243: 35–74.

Eernisse, D.J., Albert, J.S., and Anderson, F.E. 1992. Annelida andArthropoda are not sister taxa: a phylogenetic analysis ofspiralian metazoan morphology. Syst. Biol. 41: 305–330.

Ehlers, U. 1985. Das Phylogenetische System der Plathelminthes.Gustav Fischer, Stuttgart and New York.

Ehlers, U. 1986. Comments on a phylogenetic system of thePlatyhelminthes. Hydrobiologia, 132: 1–12.

Ehlers, U. 1992. On the fine structure of Paratomella rubra Rieger& Ott (Acoela) and the position of the taxon Paratomella Dörjes

in a phylogenetic system of the Acoelomorpha (Plathelminthes).Microfauna Mar. 7: 265–293.

Falciani, F., Hausdorf, B., Schroder, R., Akam, M., Tautz, D.,Denell, R., and Brown, S. 1996. Class 3 Hox genes in insectsand the origin of zen. Proc. Natl. Acad. Sci. U.S.A. 93: 8479–8484.

Felsenstein, J. 1978. Cases in which parsimony or compatibilitymethods will be positively misleading. Syst. Zool. 27: 401–410.

Field, K.G., Olsen, G.J., Lane, D.J., Giovannoni, S.J., Ghiselin,M.T., Raff, E.C., Pace, N.R., and Raff, R.A. 1988. Molecularphylogeny of the animal kingdom. Science (Wash., D.C.), 239:748–753.

Garcia-Fernandez, J., and Holland, P.W.H. 1994. Archetypal orga-nization of the amphioxus Hox gene cluster. Nature (Lond.),370: 563–566.

Gegenbaur, C. 1859. Grundzuge der vergleichenden Anatomie. W.Engelmann, Leipzig, Germany.

Giesa, S. 1966. Die Embryonalentwicklung von Monocelis fusca(Turbellaria, Proseriata). Z. Morphol. Oekol. Tiere, 57: 137–230.

Giribet, G. 2002. Current advances in the phylogenetic reconstruc-tion of metazoan evolution. A new paradigm for the Cambrianexplosion?. Mol. Phylogenet. Evol. 24: 345–357.

Giribet, G., Distel, D.L., Polz, M., Sterrer, W., and Wheeler, W.C.2000. Triploblastic relationships with emphasis on the Acoelo-mates and the position of Gnathostomulida, Cycliophora,Plathelminthes, and Chaetognatha: a combined approach of 18SrDNA sequences and morphology. Syst. Biol. 49: 539–562.

Graff, L.V. 1882. Monographie der Turbellarien. I. Rhabdocoela.Verlag W. Engelmann, Leipzig, Germany.

Graybeal, A. 1998. Is it better to add taxa or characters to a diffi-cult phylogenetic problem? Syst. Biol. 47: 9–17.

Haeckel, E. 1874. The gastraea-theory, the phylogenetic classifica-tion of the animal kingdom and the homology of the germlamellae. Q. J. Microsc. Sci. 14: 142–165.

Halanych, K.M., Bachelor, J., Aguinaldo, A.M.A., Liva, S., Hillis,D.M., and Lake, J.A. 1995. 18S rDNA evidence that the lopho-phorates are protostome animals. Science (Wash., D.C.), 267:1641–1643.

Haszprunar, G. 1996a. Plathelminthes and Plathelminthomorpha —paraphyletic taxa. J. Zool. Syst. Evol. Res. 34: 41–48.

Haszprunar, G. 1996b. The Mollusca: coelomate turbellarians ormesenchymate annelids? In Origin and evolutionary radiationsof the mollusca. Edited by J. Taylor. Oxford University Press,Oxford. pp. 1–28.

Henry, J.Q., Martindale, M.Q., and Boyer, B.C. 2000. The uniquedevelopmental program of the acoel flatworm, Neochildia fusca.Dev. Biol. 220: 285–295.

Hillis, D.M. 1999. SINEs of the perfect character. Proc. Natl.Acad. Sci. U.S.A. 96: 9979–9981.

Hochberg, R., and Litvaitis, M.K. 2001. Macrodasyda (Gastro-tricha): a cladistic analysis of morphology. Invertebr. Biol. 120:124–135.

Holland, P.W.H. 1998. Major transitions in animal evolution: a de-velopmental genetic perspective. Am. Zool. 38: 829–842.

Holland, P.W.H., and Garcia-Fernandez, J. 1996. Hox genes andchordate evolution. Dev. Biol. 173: 382–395.

Hooge, M.D., Haye, P.A., Tyler, S., Litvaitis, M.K., Kornfeld, I.2002. Molecular systematics of the Acoela (Acoelomorpha,Platyhelminthes) and its concordance with morphology. Mol.Phylogenet. Evol. 24: 333–342.

Hyman, L.H. 1951. The invertebrates. Vol. II. Platyhelminthes andRhynchocoela. McGraw-Hill Book Co., New York.

© 2004 NRC Canada

190 Can. J. Zool. Vol. 82, 2004

© 2004 NRC Canada

Baguñà and Riutort 191

Jägersten, G. 1955. On the early phylogeny of the Metazoa: thebilaterogastraea theory. Zool. Bidr. Upps. 30: 321–354.

Janicki, C. 1920. ‘Grundlinien einer Cercomer Theorie’ zurMorphologie der Trematoden und Cestoden. Festschr. Zschokke.30: 1–22.

Jenner, R.A. 2000. Evolution of animal body plans: the role ofmetazoan phylogeny at the interface between pattern and pro-cess. Evol. Dev. 2: 208–221.

Jenner, R.A., and Schram, F.R. 1999. The grand game of metazoanphylogeny: rules and strategies. Biol. Rev. (Camb.), 74: 121–142.

Joffe, B.I., and Kornakova, E.E. 1998. Notentera ivanovi Joffe etal. 1997: a contribution to the question of phylogenetic relation-ships between ‘turbellarians’ and the parasitic Platyhelminthes(Neodermata). Hydrobiologia, 383: 245–250.

Joffe, B.I., and Kornakova, E.E. 2001. Flatworm phylogeneticist:between a molecular hammer and a morphological anvil. In Theinterrelationships of the Platyhelminthes. Edited by D.T.J.Littlewood and R.D. Bray. Taylor & Francis, London, England.pp. 279–291.

Jondelius, U. 1998. Flatworm phylogeny from partial 18S rDNAsequences. Hydrobiologia, 383. 147–154.

Jondelius, U., Norén, M., and Hendelberg, J. 2001. The Prolecitho-phora. In The interrelationships of the Platyhelminthes. Editedby D.T.J. Littlewood and R.D. Bray. Taylor & Francis, London,England. pp. 74–80.

Jondelius, U., Ruiz-Trillo, I., Baguñà, J., and Riutort, M. 2002.The Nemertodermatida are basal bilaterians not members ofPlatyhelminthes. Zool. Scr. 31: 201–215.

Karling, T.G. 1940. Zur Morphologie und Systematik der Alloeo-coela Cumulata and Rhabdocoela Lecithophora (Turbellaria).Acta Zool. Fenn. 26: 1–260.

Karling, T.G. 1974. On the anatomy and affinities of theturbellarian orders. In Biology of the Turbellaria. Edited byN.W. Riser and M.P. Morse. McGraw-Hill, New York. pp. 1–16.

Katayama, T., Yamamoto, M., Wada, H., and Satoh, N. 1993.Phylogenetic position of Acoel turbellarians inferred from par-tial 18S rDNA sequences. Zool. Sci. (Tokyo), 10: 529–536.

Katayama, T., Nishioka, M., and Yamamoto, M. 1996. Phylogen-etic relationships among turbellarian orders inferred from 18SrDNA sequences. Zool. Sci. (Tokyo), 13: 747–756.

Kimmel, C.B. 1996. Was Urbilateria segmented?. Trends Genet.12: 329–331.

Knauss, E.B. 1979. Indication of an anal pore in Gnathostomulida.Zool. Scr. 8: 181–186.

Kornakova, E.E., and Joffe, B.I. 1999. A new variant of theneodermatan-type spermiogenesis in a parasitic ‘turbellarian’,Notentera ivanovi (Platyhelminthes, Seriata). Acta Zool. (Stockh.),80: 135–151.

Kristensen, R.M., and Funch, P. 2000. Micrognathozoa: a newclass with complicated jaws like those of Rotifera and Gnath-ostomulida. J. Morphol. 246: 1–49.

Ladurner, P., Rieger, R.M., and Baguñà, J. 2000. Spatial distribu-tion and differentiation potential of stem cells in hatchlings andadults in the marine platyhelminth Macrostomum sp.: a bromo-deoxyuridine analysis. Dev. Biol. 226: 231–241.

Le, T.H., Blair, D., Agatsuma, T., Humair, P.F., Campbell, N.J.,Iwagami, M., Littlewood, D.T.J, Peacock, B., Johnston, D.A.,Bartley, J., Rollinson, D., Herniou, E.A., Zarlenga, D.S., andMcManus, D.P. 2000. Phylogenies inferred from mitochondrialgene orders — a cautionary tale from the parasitic flatworms.Mol. Biol. Evol. 17: 1123–1125.

Littlewood, D.T.J., and Bray, R.A. 2001. The interrelationships of

the Platyhelminthes. Taylor & Francis, London, England. pp. 1–356.

Littlewood, D.T.J., and Olson, P.D. 2001. Small subunit rDNA andthe Platyhelminthes: signal, noise and compromise. In The inter-relationships of the Platyhelminthes. Edited by D.T.J. Littlewoodand R.D. Bray. Taylor & Francis, London, England. pp. 262–278.

Littlewood, D.T.J., Rohde, K., and Clough, K.A. 1999a. The inter-relationships of all major groups of Platyhelminthes: phylogen-etic evidence from morphology and molecules. Biol. J. Linn.Soc. 66: 75–114.

Littlewood, D.T.J., Rohde, K., Bray, R.A., and Herniou, E.A.1999b. Phylogeny of the Platyhelminthes and the evolution ofparasitism. Biol. J. Linn. Soc. 68: 257–287.

Littlewood, D.T.J., Curini-Galletti, M., and Herniou, E.A. 2000.The interrelationships of Proseriata (Platyhelminthes: Seriata)tested with molecules and morphology. Mol. Phylogenet. Evol.16: 449–466.

Littlewood, D.T.J., Olson, P.D., Telford, M.J., Herniou, E.A., andRiutort, M. 2001a. Elongation Factor 1 – alpha sequences alonedo not assist in resolving the position of the Acoela within theMetazoa. Mol. Biol. Evol. 18: 437–442.

Littlewood, D.T.J., Cribb, T.H., Olson, P.D., and Bray, R.A. 2001b.Platyhelminth phylogenetics — a key to understanding parasit-ism? Belg. J. Zool. 131(Suppl. 1): 35–46.

Litvaitis, M.K., and Newman, L.J. 2001. A molecular frameworkfor the phylogeny of the Pseudocerotidae (Platyhelminthes,Polycladida). Hydrobiologia, 444: 177–182.

Litvaitis, M.K., and Rohde, K. 1999. A molecular test ofplatyhelminth phylogeny: inferences from partial 28S rDNA se-quences. Invertebr. Biol. 118: 42–56.

Litvaitis, M.K., Curini-Galletti, M., Martens, and P.M., Kocher,T.D. 1996. A reappraisal of the systematics of the Monoceli-didae (Platyhelminthes, Proseriata) — inferences from rDNA se-quences. Mol. Phylogenet. Evol. 6: 150–156.

Llevellyn, J. 1965. The evolution of parasitic platyhelminths. InThird Symposium of the British Society for Parasitology. Editedby A. Taylor. Blackwell Scientific Publications, Oxford. pp. 47–78.

Lockyer, A.E., Olson, P.D., and Littlewood, D.T.J. 2003. Utility ofcomplete large and small subunit rRNA genes in resolving thephylogeny of the Neodermata (Platyhelminthes): implicationsand a review of the cercomer theory. Biol. J. Linn. Soc. 78:155–171.

Miyamoto, M.M. 1999. Molecular systematics: Perfect SINEs ofevolutionary history? Curr. Biol. 9: 1–816.

Mollaret, I., Jamieson, B.G.M., Adlard, R.D., Hugall, A.,Lecointre, G., Chombard, C., and Justine, J.-L. 1997. Phylogen-etic analysis of the Monogenea and their relationships withDigenea and Eucestoda inferred from 28S rDNA sequences.Mol. Biochem. Parasitol. 90: 433–438.

Nielsen, C. 2001. Animal evolution: interrelationships of the livingphyla. 2nd ed. Oxford University Press, Oxford.

Nielsen, C., and Norrevang, A. 1985. The trochaea theory: an ex-ample of life cycle phylogeny. In The origins and relationshipsof lower invertebrates. Edited by S. Conway Morris., J.D.George., R. Gibson, and H.M. Platt. Clarendon Press, Oxford.pp. 28–41.

Nikaido, M., Rooney, A.P., and Okada, N. 1999. Phylogenetic rela-tionships among cetartiodactyls based on insertions of short andlong interpersed elements: hippopotamuses are the closest extantrelatives of whales. Proc. Natl. Acad. Sci. U.S.A. 96: 10 261 –10 266.

Nogi, T., and Watanabe, K. 2001. Position-specific and non-

colinear expression of the planarian posterior (abdominal-B-like)gene. Dev. Growth Differ. 43: 177–184.

Norén, M., and Jondelius, U. 2002. The phylogenetic position ofthe Prolecithophora (Rhabditophora, ‘Platyhelminthes’). Zool.Scr. 31: 403–414.

Orii, H., Kato, K., Umesono, Y., Sakurai, T., Agata, K., andWatanabe, K. 1999. The planarian HOM/HOX homeobox genes(Plox) expressed along the anteroposterior axis. Dev. Biol. 210:456–468.

Pasquinelli, A.E., McCoy, A., Jimenez, E., Saló, E., Ruvkun, G.,Martindale, M.Q., and Baguñà, J. 2003. Expression of the 22nucleotide let-7 heterochronic RNA throughout the Metazoa: arole in life history evolution? Evol. Dev. 5: 372–378.

Peterson, K.J., and Eernisse, D.J. 2001. Animal phylogeny and theancestry of bilaterians: inferences from morphology and 18SrDNA gene sequences. Evol. Dev. 3: 170–205.

Peterson, K.J., Cameron, R.A., and Davidson, E.H. 2000.Bilaterian origins: significance of new experimental observa-tions. Dev. Biol. 219: 1–17.

Philippe, H., Chenuil, A., and Adoutte, A. 1994. Can the Cambrianexplosion be inferred through molecular phylogeny? Develop-ment (Camb.), Suppl.: 15–25.

Raikova, O.I., Reuter, M., Jondelius, U., and Gustafsson, M.K.S.2000. The brain of the Nemertodermatida (Platyhelminthes) asrevealed by anti-5HT and anti-FMRFamide immunostainings.Tissue Cell, 32: 358–365.

Raikova, O.I., Reuter, M., and Justine, J-L. 2001. Contributions tothe phylogeny and systematics of the Acoelomorpha. In The in-terrelationships of the Platyhelminthes. Edited by D.T.J.Littlewood and R.D. Bray. Taylor & Francis, London, England.pp. 13–23.

Regier, J.C., and Shultz, J.W. 2001. Elongation factor-2: a usefulgene for arthropod phylogenetics. Mol. Phylogenet. Evol. 20:136–148.

Remane, A. 1963. The enterocoelic origin of the coelom. In Thelower Metazoa. Edited by E.C. Dougherty. University of Califor-nia Press, Berkeley. pp. 78–90.

Remane, A., Storch, V., and Welsch, U. 1980. SystematischeZoologie. Gustav Fischer, Stuttgart, Germany.

Reuter, M., Raikova, O.I., and Gustafsson, M.K. 1998. An endo-crine brain? The pattern of FMRFamide immunoreactivity inAcoela (Platyhelminthes). Tissue Cell, 30: 57–63.

Rieger, R. 1985. The phylogenetic status of the acoelomate organi-sation within the Bilateria: a histological perspective. In The ori-gin and relationships of lower invertebrates. Edited by S.Conway Morris, R. Gibson, and H.M. Platt. Oxford UniversityPress, Oxford. pp. 101–122.

Rieger, R.M. 1986. Über dem Ursprung der Bilateria: dieBedeutung der Ultrastrukturforschung für eines neues Verstehender Metazoenevolution. Verh. Dtsch. Zool. Ges. 79: 31–50.

Rieger, R.M. 1994. The biphasic life cycle — a central theme ofmetazoan evolution. Am. Zool. 34: 484–491.

Rieger, R.M. 2001. Phylogenetic systematics of the Macrosto-morpha. In The interrelationships of the Platyhelminthes. Editedby D.T.J. Littlewood and R.D. Bray. Taylor & Francis, London,England. pp. 28–38.

Rieger, R.M., and Ladurner, P. 2001. Searching for the stem spe-cies of the Bilateria. Belg. J. Zool. 131(Suppl. 1): 27–34.

Riutort, M., Field, K.G., Turbeville, J.M., Raff, R.R., and Baguñà,J. 1992. Enzyme electrophoresis, 18S rRNA sequences, and lev-els of phylogenetic resolution among several species of freshwa-ter planarians (Platyhelminthes, Tricladida, Paludicola). Can. J.Zool. 70: 1425–1429.

Riutort, M., Field, K.G., Raff, R.R., and Baguñà, J. 1993. 18SrRNA sequences and phylogeny of Platyhelminthes. Biochem.Syst. Ecol. 21: 71–77.

Rohde, K. 1990. Phylogeny of Platyhelminthes, with special refer-ence to parasitic groups. Int. J. Parasitol. 20: 979–1007.

Rohde, K. 1997. The origins of parasitism in the Platyhelminthes;a summary interpreted on the basis of recent literature. Int. J.Parasitol. 27: 739–746.

Rohde, K. 2001. The Aspidogastrea: an archaic group of Platy-helminthes. In The interrelationships of the Platyhelminthes.Edited by D.T.J. Littlewood and R.D. Bray. Taylor & Francis,London, England. pp. 159–167.

Rohde, K., Hefford, C., Ellis, J.T., Baverstock, P.R., Johnson,A.M., Watson, N.A., and Dittmann, S. 1993. Contributions tothe phylogeny of Platyhelminthes based on partial sequencing of18S ribosomal DNA. Int. J. Parasitol. 23: 705–724.

Rohde, K., Hayward, C., and Heap, M. 1995. Aspects of the ecol-ogy of metazoan ectoparasites of marine fishes. Int. J. Parasitol.25: 945–970.

Rokas, A., and Holland, P.W. 2000. Rare genomic changes as atool for phylogenetics. Trends Ecol. Evol. 15: 454–459.

Ruiz-Trillo, I., Riutort, M., Littlewood, D.T.J., Herniou, E.A., andBaguñà, J. 1999. Acoel flatworms: earliest extant BilaterianMetazoans, not members of Platyhelminthes. Science (Wash.,D.C.), 283: 1919–1923.

Ruiz-Trillo, I., Paps, J., Loukota, M., Ribera, C., Jondelius, U.,Baguñà, J., and Riutort, M. 2002. A phylogenetic analysis ofmyosin heavy chain type II sequences corroborates that Acoelaand Nemertodermatida are basal bilaterians. Proc. Natl. Acad.Sci. U.S.A. 99: 11 246 – 11 251.

Ruiz-Trillo, I., Riutort, M., Fourcade, H.M., Baguñà, J., and Boore,J.L. 2004. Mitochondrial genome data support the basal positionof Acoelomorpha and the polyphyly of the Platyhelminthes.Mol. Phylogenet. Evol. In press.

Saló, E., Tauler, J., Jiménez, E., Bayascas, J.R., Gonzalez-Linares,J., García-Fernández, J., and Baguñà, J. 2001. Hox and ParaHoxgenes in flatworms. Characterization and expression. Am. Zool.41: 652–663.

Sluys, R. 1989. Phylogenetic relationships of the triclads (Platy-helminthes, Seriata, Tricladida). Bijdr. Dierkd. 59: 3–25.

Sluys, R. 1990. A monograph of the Dimarcusidae (Platyhel-minthes, Seriata, Tricladida). Zool. Scr. 19: 13–29.

Smith, J.P.S., III., and Tyler, S. 1985. The acoel turbellarians: king-pins of metazoan evolution or a specialized offshot?. In Theorigins and relationships of lower invertebrates. Edited by S.Conway-Morris, J.D. George, R. Gibson, and H.M. Platt. Clar-endon Press, Oxford. pp. 123–142.

Smith, J.P.S., III., Tyler, S., and Rieger, R.M. 1986. Is theTurbellaria polyphyletic? Hydrobiologia, 132: 71–78.

Sogin, M.L. 1991. Early evolution and the origin of eukaryotes.Curr. Opin. Genet. Dev. 1: 457–463.

Sopott-Ehlers, B. 1985. The phylogenetic relationships within theSeriata (Platyhelminthes). In The origins and relationships oflower invertebrates. Edited by S. Conway-Morris, J.D. George,R. Gibson, and H.M. Platt. Clarendon Press, Oxford. pp. 159–167.

Telford, M.J. 2000. Turning Hox “signatures” into synapomor-phies. Evol. Dev. 2: 360–364.

Telford, M.J. 2001. Embryology and developmental genes as cluesto flatworm relationships. In The interrelationships of the platy-helminthes. Edited by D.T.J. Littlewood and R.D. Bray. Taylor& Francis, London, England. pp 257–261.

Telford, M.J., Herniou, E.A., Russell, R.B., and Littlewood, D.T.2000. Changes in mitochondrial genetic codes as phylogenetic

© 2004 NRC Canada

192 Can. J. Zool. Vol. 82, 2004

characters: two examples from the flatworms. Proc. Natl. Acad.Sci. U.S.A. 97: 11 359 – 11 364.

Telford, M.J., Lockyer, A.E., Cartwright-Finch, C., and Littlewood,D.T.J. 2003. Combined large and small subunit ribosomal RNAphylogenies support a basal position of the acoelomorph flat-worms. Proc. R. Soc. Lond. B Biol. Sci., 270: 1077–1083.

Tyler, S. 2001. The early worm: Origins and relationships of thelower flatworms. In The interrelationships of the platyhelmin-thes. Edited by D.T.J. Littlewood and R.D. Bray. Taylor & Fran-cis, London, England. pp. 3–12.

Tyler, S., and Tyler, M.S. 1997. Origin of the epidermis in parasiticPlatyhelminthes. Int. J. Parasitol. 27: 715–738.

Tyler, S., and Bush, L.F. 2003. Turbellarian taxonomic database.Version 1.0. Available from http://devbio.umesci.maine.edu/styler/turbellaria [accessed 18–19 June 2003].

Wilkinson, M., Thorley, J.L., Littlewood, D.T.J., and Bray, R.A.2001. Towards a phylogenetic supertree of Platyhelminthes? InThe Interrelationships of the Platyhelminthes. Edited by D.T.J.Littlewood and R.D. Bray. Taylor & Francis, London, England.pp. 292–301.

Willmer, P. 1990. Invertebrate relationships. Patterns in animalevolution. Cambridge University Press, Cambridge, UK.

Woese, C.R. 1987. Bacterial evolution. Microbiol. Rev. 51: 221–271.

Woese, C.R., Magrum, L.J., and Fox, G.E. 1978. Archaebacteria. J.Mol. Evol. 11: 245–251.

Zamparo, D., Brooks, D.R., Hoberg, E.P., and McLennan, D.A.2001. Phylogenetic analysis of the Rhabdocoela (Platyhelmin-thes) with emphasis on the Neodermata and relatives. Zool. Scr.30: 59–77.

Zrzavy, J. 2001. The interrelationships of metazoan parasites: a re-view of phylum- and higher-level hypotheses from recent mor-phological and molecular phylogenetic analyses. Folia Parasitol.(Prague), 48: 81–103.

Zrzavy, J. 2003. Gastrotricha and metazoan phylogeny. Zool. Scr.32: 61–82.

Zrzavy, J., Milhulka, S., Kepka, P., Bezdek, A., and Tietz, D. 1998.Phylogeny of the Metazoa based on morphological and 18S ri-bosomal DNA evidence. Cladistics, 14: 249–285.

Zwickl, D.J., and Hillis, D.M. 2002. Increased taxon samplinggreatly reduces phylogenetic error. Syst. Biol. 51: 588–598.

© 2004 NRC Canada

Baguñà and Riutort 193