The Nemertodermatida are basal bilaterians and not members of the Platyhelminthes Blackwell Science Ltd ULF JONDELIUS, IÑAKI RUIZ-TRILLO, JAUME BAGUÑÀ & MARTA RIUTORT Accepted: 19 September 2001 Jondelius, U., Ruiz-Trillo, I., Baguñà, J. & Riutort, M. (2002). The Nemertodermatida are basal bilaterians and not members of the Platyhelminthes. Zoologica Scripta, 31, 201–215. Recent hypotheses on metazoan phylogeny have recognized three main clades of bilaterian animals: Deuterostomia, Ecdysozoa and Lophotrochozoa. The acoelomate and ‘pseudocoelomate’ metazoans, including the Platyhelminthes, long considered basal bilaterians, have been referred to positions within these clades by many authors. However, a recent study based on ribosomal DNA placed the flatworm group Acoela as the sister group of all other extant bilaterian lineages. Unexpectedly, the nemertodermatid flatworms, usually considered the sister group of the Acoela together forming the Acoelomorpha, were grouped separately from the Acoela with the rest of the Platyhelminthes (the Rhabditophora) within the Lophotrochozoa. To re-evaluate and clarify the phylogenetic position of the Nemertodermatida, new sequence data from 18S ribosomal DNA and mitochondrial genes of nemertodermatid and other bilaterian species were analysed with parsimony and maximum likelihood methods. The analyses strongly support a basal position within the Bilateria for the Nemertodermatida as a sister group to all other bilaterian taxa except the Acoela. Despite the basal position of both Nemertodermatida and Acoela, the clade Acoelomorpha was not retrieved. These results imply that the last common ancestor of bilaterian metazoans was a small, benthic, direct developer without segments, coelomic cavities, nephrida or a true brain. The name Nephrozoa is proposed for the ancestor of all bilaterians excluding the Nemertodermatida and the Acoela, and its descendants. Ulf Jondelius, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, SE-752 36 Uppsala, Sweden. E-mail: ulf.jondelius@ebc.uu.se Iñaki Ruiz-Trillo, Jaume Baguñà & Marta Riutort, Departament de Genetica, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain Introduction Platyhelminth flatworms have often played a crucial role in discussions of metazoan phylogeny as the sister group or even ‘ancestors’ of other Bilateria (e.g. Willmer 1990). Although monophyly of the Platyhelminthes has long been assumed, the relationships between the three major platyhelminth clades, Acoelomorpha ( Nemertodermatida + Acoela), Catenulida and Rhabditophora are controversial. Difficulties identifying robust morphological synapomorphies uniting Rhabditophora with Catenulida and with the Acoelomorpha led Smith et al. (1986) to question the monophyly of the Platyhelminthes, although they did not provide an alternative hypothesis for the sister groups of the three clades. A critical reassessment of morphological evidence within the context of metazoan evolution led Haszprunar (1996) to consider Platyhelminthes a paraphyletic group, with Acoelomorpha being the earliest bilaterian offshoot followed by the Rhabditophora, the Catenulida and the rest of the Bilateria. Another analysis of metazoan phylogeny using a large set of morphological characters in combination © The Norwegian Academy of Science and Letters • Zoologica Scripta, 31, 2, April 2002, pp201 – 215 with 18S ribosomal DNA (rDNA), also indicated the Platyhelminthes to be a paraphyletic assemblage (Zrzavy et al. 1998). However, in most zoology textbooks, flatworms are still featured as a monophyletic group. Recent analyses of molecular data have challenged the longheld position of Platyhelminthes as ancestral bilaterians as well as flatworm monophyly. 18S rDNA sequence data and Hox gene molecular signatures form the basis for the recognition of three main clades of bilaterian animals: Deuterostomia, Ecdysozoa and Lophotrochozoa (e.g. Aguinaldo et al. 1997; Adoutte et al. 1999). The acoelomate and ‘pseudocoelomate’ metazoans, including the bulk of the Platyhelminthes (the Rhabditophora, or probably Rhabditophora + Catenulida), are now referred to positions within these clades by many authors (Balavoine 1997, 1998; Carranza et al. 1997; Bayascas et al. 1998; Littlewood et al. 1999; De Rosa et al. 1999; Adoutte et al. 2000; Saló et al. 2001); but see Giribet et al. (2000) for a different view. Thus, Platyhelminthes are now generally considered lophotrochozoans. However, a recent study based on 201 Nemertodermatida are basal bilaterians • U. Jondelius et al. rDNA placed the flatworm group Acoela as the sister group of all other extant bilaterian lineages (Ruiz-Trillo et al. 1999) and separated it from the Rhabditophora which fell within the Lophotrochozoa. Surprisingly, the nemertodermatid flatworms, usually considered the sister group of the Acoela ( Ehlers 1985), were grouped inside the rest of the Platyhelminthes (the Rhabditophora) within the Lophotrochozoa, and separately from the acoels. This conflicted with perceived morphological synapomorphies uniting the Acoela and Nemertodermatida into the clade Acoelomorpha ( Ehlers 1985; Smith et al. 1986) and with apomorphies shared by the Rhabditophora but absent from Nemertodermatida. The known Nemertodermatida comprise 10 –11 species of small marine worms. When the first representative of the group was formally described, Steinböck (1930 –31) regarded it as the ‘most primitive turbellarian’ and classified it within the Acoela. In his description of the second nemertodermatid, Westblad (1937) noted some important differences between the Nemertodermatida and the Acoela. For example, the presence of a permanent intestinal cavity in the former. These features formed the basis for the erection of a separate taxon outside the Acoela, the Nemertodermatida ( Karling 1940). The anatomical simplicity of the nemertodermatids was noted by Karling, who considered the Nemertodermatida to be closest to the ‘turbellarian archetype’ in his phylogenetic analysis of the free-living flatworms ( Karling 1974). Later, ultrastructural studies of Nemertodermatida and Acoela revealed similarities in their epidermal cilia interpreted as synapomorphies of the two taxa [Tyler & Rieger 1977; Ehlers 1985; Smith & Tyler 1985; reviewed in Lundin (1997, 1998)], and the taxon Acoelomorpha ( Ehlers 1985) was recognized on this basis, with Acoela and Nemertodermatida as sister groups. The first molecular studies placing the Nemertodermatida within the Rhabditophora, separately from the acoels, were based on one 18S rDNA sequence from a single nemertodermatid species (Carranza et al. 1997; Littlewood et al. 1999). When the position of the Acoela within the Metazoa was tested using the relative rate test ( Wilson et al. 1977) to eliminate putatively fast evolving 18S rDNA, nemertodermatids again fell within the Rhabditophora and separate from the Acoela, which branched as the first bilaterians ( Ruiz-Trillo et al. 1999). This led some workers to question the separation of the Nemertodermatida from the Acoela, suggesting that the early branching of the Acoela was erroneous, and the position of the Nemertodermatida within the Lophotrochozoa was also indicative of acoel relationships (Adoutte et al. 2000; Peterson et al. 2000; Dewel 2000; Telford 2001). This argument seemed to be reinforced by analyses of elongation factor 1-alpha (EF1-α) from a single species of the Acoela, which placed the group close to the Tricladida, a derived group within the Rhabditophora (Berney et al. 2000). However, new EF1-α sequences from additional species of Acoela, 202 Platyhelminthes and other Metazoa do not support a Tricladida– Acoela clade (Littlewood et al. 2001). Moreover, a number of morphological synapomorphies present in Tricladida and other Rhabditophora, but missing in the Acoela (e.g. heterocellular female gonads, rhabdites, protonephridia), conflict with a position of the Acoela within the Rhabditophora. The phylogenetic position of the Nemertodermatida needs to be carefully re-evaluated. Some morphological characters seem to link them to the acoels, but a position among the Rhabditophora within the Lophotrochozoa, as indicated by previous molecular studies (Carranza et al. 1997; Littlewood et al. 1999), appears unlikely. Therefore, it is important to know whether nemertodermatids join the acoels as the earliest known extant bilaterians separate from the rest of the Platyhelminthes or whether they are members of the Platyhelminthes within the Lophotrochozoa. In this paper, 18S rDNA and mitochondrial nucleotide sequences from three nemertodermatid species were obtained and used to reconstruct the phylogenetic position of the Nemertodermatida. Additional species of acoels and rhabditophorans, as well as a large data set of metazoan sequences, were also included in the analyses. Materials and methods Organisms Three representatives of the Nemertodermatida and one of the Acoela were used to obtain sequences from the 18S rRNA gene. One species of Nemertodermatida and one rhabditophoran were used to obtain sequences from the mitochondrial genes COI and Cytb (Table 1). The new 18S rDNA sequences were compared with a large set of 18S rDNA sequences from GenBank representing all major metazoan groups (Table 2). DNA extraction, amplification and sequencing DNA extraction, polymerase chain reaction amplification and sequencing of 18S rDNA were carried out as described in Norén & Jondelius (1999). For mitochondrial genes, DNA was extracted using the Qiamp DNA Mini Kit (Qiagen Inc.,Valencia, CA, USA). A 700-bp fragment of the COI (Cytochrome Oxidase c.: subunit I) gene and 450 bp of the Cytb (Cytochrome b) gene were amplified using universal primers. For Cytb the primers used were: cytb424–444: 5′-ggW TAY gTW YTW CCW TgR ggW CAR AT-3′ and cytb876–847: 5′-gCR TAW gCR AAW ARR AAR TAY CAY TCW gg-3′ (primer sequences provided by Dr Jeff Boore). The primers used to amplify COI were: LCO1490: 5′-ggt caa caa atc ata aag ata ttg g-3′ and HCO2198: 5′-taa act tca ggg tga cca aaa aat c-3′ (Folmer et al. 1994). Sequence alignment and phylogenetic analyses The 18S rDNA sequences were aligned according to a secondary structure model (Gutell et al. 1985). Positions that Zoologica Scripta, 31, 2, April 2002, pp201– 215 • © The Norwegian Academy of Science and Letters U. Jondelius et al. • Nemertodermatida are basal bilaterians Table 1 Taxa sequenced for this study and GenBank Accession nos. Taxon Nemertodermatida Meara stichopi Nemertoderma bathycola Nemertoderma westbladi Nemertoderma westbladi Nemertoderma westbladi Acoela Paraphanostoma cycloposthium Paratomella rubra Paratomellla rubra Simplicomorpha gigantorhabditis Rhabditophora Microstomum lineare Microstomum lineare Gene GenBank entry Collection locality 18S 18S 18S COI Cytb AF327724 AF327725 AF327726 AF329183 AF329184 Espegrend, Norway Kristineberg, Sweden Kristineberg, Sweden Kristineberg, Sweden Kristineberg, Sweden 18S COI Cytb Cytb AF329178 AF329179 AF329180 GenBank Accession no. Kristineberg, Sweden Barcelona, Spain Barcelona, Spain COI Cytb AF329181 AF329182 Turku, Finland Turku, Finland Table 2 Terminals included in the analyses with GenBank Accession nos (sequences reported in this paper are in bold). Terminals marked with * were used only in the parsimony analysis. Terminals marked with § were used only in the maximum likelihood analyses. Unmarked terminals were used in both analyses. Higher taxon Terminal Accession no. Acoela Paratomella rubra Symsagittifera psammophila* Simplicomorpha gigantorhabditis* Atriofonta polyvacuola* Paedomecynostomum bruneum* Philomecynostomum lapillum* Anapaerus tvaerminnensis* Postmecynostomum pictum* Haplogonaria syltensis* Actinoposthia beklemischevi* Amphiscolops sp.* Convoluta convoluta* Acoel sp.3* Acoel sp.2* Anaperus biaculeatus* Aphanostoma virescens* Childia groenlandica* Symsagittifera roscoffensis* Convoluta pulchra* Praesagittifera naikaiensis* Polycelis nigra* Dendrocoelum lacteum* Crenobia alpina* Discocelis tigrina Planocera multitentaculata* Geocentrophora sp. Macrostomum tuba Microstomum lineare Monocelis lineata Archiloa ribularis* Urastoma cyprinae* Echinococcus granulosus* Schistosoma mansoni Fasciolopsis bushi Gylaunchen sp.* AF102892 AF102893 AF102894 AF102895 AF102896 AF102897 AF102898 AF102899 AF102900 AJ012522 AJ012523 AJ012524 AJ012525 AJ012526 AJ012527 AJ012528 AJ012529 AJ012530 U70086 D83381 AF013151 M58346 M58345 U70074 D17562 U70080 U70081 U70083 U45961 U70077 AF167422 U27015 M62652 L06668 L06669 Rhabditophora Higher taxon Catenulida Nemertodermatida Deuterostomia Hemichordata Echinodermata Chordata Lophotrochozoa Mollusca Annelida © The Norwegian Academy of Science and Letters • Zoologica Scripta, 31, 2, April 2002, pp201 – 215 Terminal Accession no. Lobatostoma manteri* Neomicrocotyle pacifica* Stenostomum leucops Suomina sp. Nemertinoides elongatus* Meara sp.* Meara stichopi* Nemertoderma bathycola Nemertoderma westbladi L16911 AJ228787 U70085 AJ012532 U70084 AF051328 AF327724 this study AF327725 this study AF327726 this study Balanoglossus carnosus Saccoglossus kowalewskii Antedon serrata Ophioplocus japonicus Amphipholis squamata* Asterias amurensis* Branchiostoma floridae§ Lampetra aepyptera§ Xenopus laevis§ Mus musculus§ D14359 L28054 D14357 D14361 X97156 D14358 M97571 M97573 X04025 X00686 Acanthopleura japonica Lepidochitona corrugata Argopecten irradians Chlamys islandica Nerita albicilla Limicolaria kambeul Antalis vulgaris* Liolophura japonica* Monodonta labio* Eisenia foetida Enchytraeus sp. Hirudo medicinalis Haemopis sanguisuga Lanice conchilega X70210 X91975 L11265 L11232 X91971 X60374 X91980 X76210 X94271 X79872 U95948 Z83752 X91401 X79873 203 Nemertodermatida are basal bilaterians • U. Jondelius et al. Table 2 continued Higher taxon Nemertini Sipuncula Brachiopoda Entoprocta Bryozoa Phoronida Echiura Pogonophora Ecdysozoa Tardigrada Arthropoda Priapulida Terminal Accession no. Higher taxon Terminal Accession no. Nereis virens Glycera americana* Lineus sp. Prostoma eilhardi§ Phascolosoma granulatum Terebratalia transversa Lingula lingua Barentsia hildegardae Pedicellina cernua Plumatella repens Phoronis vancouverensis Ochetostoma erythrogrammom Ridgeia piscesae Siboglinum fiordicum Z83754 U19519 X79878 U29494 X79874 U12650 X81631 AJ001734 U36273 U12649 U12648 X79875 X79877 X79876 Kinorhyncha Nematomorpha Others Gnathostomulida Rotifera Pycnophyes hielensis Gordius aquaticus* U67997 X87985 Gnathostomula paradoxa* Philodina acuticornis* Brachionus plicatilis§ Moliniformis moliniformis* Neoechynorhynchus pseudemydis* Centrorhynchus conspectus* Lepidodermella squammata Chaetonotus sp. Z81325 U91281 U49911 Z19562 U41400 U41399 U29198 AJ001735 Macrobiotus hufelandi* Odiellus troguloides Aphonopelma sp. Berndtia purpurea Panulirus argus Tenebrio molitor Polistes dominulus Scolopendra cingulata Priapulus caudatus X81442 X81441 X13457 L26511 U19182 X07801 X77785 U29493 X87984 Trichoplax adhaerens Scypha ciliata Microciona prolifera Tetilla japonica* Anemonia sulcata Antipathes galapagensis* Atolla vanhoeffeni Anthopleura kurogane* Tripedalia cystophora Beroe cucumis§ Mnemiopsis leidyi§ Saccharomyces cerevisiae§ L10828 L10827 L10825 D15067 X53498 AF100943 AF100942 Z21671 L10829 D15068 L10826 Z75578 could not be unambiguously aligned were excluded from the analysis, leaving 1655 characters in 104 taxa for the parsimony analysis and 1237 in 66 taxa for the maximum likelihood analysis (with reference to different numbers of taxa used in the analyses, see below). The mitochondrial sequences (COI and Cytb) were aligned with the software CLUSTALW (Thompson et al. 1994), based on the amino acid sequences. The alignments were then edited in the GDE software package (Smith et al. 1994). A combined 18S rDNA and mitochondrial data set was compiled from our own data and sequences available from GenBank. Because sequences for all of the genes were not available for some of the species, some terminals are a compilation of sequences from closely related species (see Table 3). In this data set, only the first and second codon positions of the mitochondrial genes were used, giving a total of 2143 characters (750 parsimony informative, 19 taxa). Long branch attraction ( LBA, also known as unequal rate artefacts) may distort the results of phylogeny reconstruction by grouping the longest branches together irrespective of the actual phylogeny ( Felsenstein 1978; Hendy & Penny 1989). Although it is difficult to demonstrate LBA with empirical data, it is theoretically possible that the phenomenon may be a concern in phylogenetic analyses that incorporate widely divergent taxa. LBA is a special case of convergence: random similarities on unrelated clades drive them together in a parsimony analysis. It is not known how large the differences 204 Acanthocephala Gastrotricha Diploblasts Placozoa Porifera Cnidaria Ctenophora Fungi in branch length have to be to produce LBA artefacts, but it is obvious that a reduction in homoplasy in the data set will reduce the risk of LBA artefacts. We used a method devised by J. S. Farris to reduce the homoplasy level in our data by recoding the nucleotide sequences into triplets: all occurring triplet combinations of nucleotides were each given their unique code ( Farris, prerelease software). Thereby the number of possible character states was increased from four to 64 and the likelihood of chance convergence was reduced [ Felsenstein’s (1978) initial simulation studies were performed using binary characters]. In an effort to avoid artefacts caused by insufficient taxonomic sampling and to break up any remaining long branches, we included a large selection of terminals from the studied groups: 21 acoel, 17 rhabditophoran and two catenulid terminals were analysed together with the four nemertodermatid sequences available and 60 other metazoans. The recoded data set consisted of 457 characters, all of them parsimony informative. The parsimony analysis ( heuristic search with 1000 random additions and the Tree Bisection and Reconnection (TBR) option enabled) was performed with PAUP* (Swofford 2000). Parsimony jackknifing of the combined 18S rDNA and mitochondrial data set (19 terminals, 2143 characters, 750 of which were parsimony informative) was performed with the XAC software ( J. S. Farris prerelease software). Five thousand jackknife replicates with nine addition replicates each were performed. Zoologica Scripta, 31, 2, April 2002, pp201– 215 • © The Norwegian Academy of Science and Letters U. Jondelius et al. • Nemertodermatida are basal bilaterians Table 3 Composition of the concatenated 18S ribosomal DNA and mitochondrial data set. Higher taxon Species 18S GenBank 18S Species mitochondrial GenBank COI GenBank Cytb Cnidaria Nemertodermatida Acoela Platyhelminthes1 Platyhelminthes2 Annelida1 Annelida2 Brachiopoda Mollusca1 Mollusca2 Mollusca3 Nematoda1 Nematoda2 Arthropoda1 Arthropoda2 Echinodermata1 Echinodermata2 Chordata1 Chordata2 Metridium sp. Nemertoderma westbladi Paratomella rubra Microstomum lineare Echinococcus granularis Lumbricus terrestris Nereis virens Terebratulina retusa Cepaea nemoralis Liolophura japonica Helix aspersa Ascaris suum Gnathostoma binucleatum Polistes dominulus Archeta domesticus Arbacia lixula Strongylocentrotus purpuratus Gallus gallus Cyprinus carpio AF052889 This study AF102892 U70082 U27015 AJ272183 Z83754 U08324 AJ224921 X70210 X91976 U94367 Z96946 X77785 X95741 X37514 L28056 AF173612 AF133089-1st AF021880-2nd M. senile N. westbladi P. rubra M. lineare E. multilocularis L. terrestris Platynereis dumerilii T. retusa C. nemoralis Katharina tunicata Albinaria coerulea A. suum Onchocerca volvulus Apis mellifera Locusta migratoria A. lixula S. purpuratus G. gallus C. carpio NC000933 This study This study This study AB018440 U24570 AF178678 AJ245743 U23045 U09810 X83390 X54253 AF015193 L06178 X80245 X80396 X12631 X52392 X61010 NC000933 This study This study This study AB018440 U24570 AF178678 AJ245743 U23045 U09810 X83390 X54253 AF015193 L06178 X80245 X80396 X12631 X52392 X61010 In the maximum likelihood analyses of the 18S rDNA sequences alone, only the 55 taxa that passed the relative rate test in our previous study (Ruiz-Trillo et al. 1999), together with nine outgroup taxa, were used (Table 2, indicated with ?). The three new nemertodermatid sequences were also submitted to the same test. Only two passed and consequently both were added to the data set, giving a total of 66 taxa. The analyses were performed with the FASTDNAML software (Olsen et al. 1994) using global rearrangements and jumble options. The categories option was used to take into account the among-site rate variation, introducing the parameters previously calculated with PUZZLE 4.0. Branch support for the ribosomal data set was calculated only for the nodes of interest, through a four-cluster likelihood mapping analysis with PUZZLE 4.0 (Strimmer & von Haeseler 1996) taking into account among-site rate variation. This approach allows an analysis of the support for internal branches in a tree without having to compute the overall tree. Every internal branch in a completely resolved tree defines up to four clusters of sequences. These four clusters can be defined in the data file and the program will build a quartet tree for each of all possible combinations of four species, always taking one from each group. The result is represented on a triangle in which each corner represents one of the three possible unrooted trees uniting the four clusters of species. The distribution of points within this triangle indicates the level of support for the internal branch under analysis. In the analysis to determine the support for the branch separating the Acoela from the rest of the Bilateria, the four clusters were: (A) fungus + diploblasts; (B) Acoela; (C) Deuterostomia; and (D) Protostomia. To determine the © The Norwegian Academy of Science and Letters • Zoologica Scripta, 31, 2, April 2002, pp201 – 215 support for the branch separating the Nemertodermatida from the rest of the Bilateria (except the Acoela), the groups were: (A) fungus + diploblasts + Acoela; (B) Nemertodermatida; (C) Deuterostomia; and (D) Protostomia. A further analysis was carried out in which a maximum likelihood tree was built without including any outgroup species, or the Acoela. In this analysis, the long branch separating diploblastic from triploblastic animals is eliminated. Consequently, one would expect the Nemertodermatida to branch close to the Platyhelminthes if their basal position was an artefact (Giribet et al. 2000) due to LBA caused by diploblasts. Conversely, if nemertodermatids, together with acoels, are basal triploblasts and the remaining platyhelminths (the Rhabditophora) are derived spiralians, nemertodermatids should not group with the Rhabditophora. The maximum likelihood analyses of the combined mitochondrial and ribosomal data set were performed as explained above. An additional maximum likelihood analysis was carried out without the most variable positions (category 8 of the gamma distribution). Branch support for this data set was calculated through quartet puzzling (50 000 replicates) with PUZZLE 4.0, taking into account among-site rate variation. Results Parsimony analysis The strict consensus tree from 52 most-parsimonious trees calculated from the triplet-coded 18S rDNA nucleotide data is shown in Fig. 1 (retention index = 0.56). The cladogram shows the Acoela as the sister group of the remaining Bilateria 205 Nemertodermatida are basal bilaterians • U. Jondelius et al. Fig. 1 Strict consensus tree of 52 most-parsimonious trees resulting from a parsimony analysis of the triplet-coded 18S ribosomal DNA data set (PAUP* 1000 random additions/TBR equal weights 457 characters, all of them parsimony informative). 206 Zoologica Scripta, 31, 2, April 2002, pp201– 215 • © The Norwegian Academy of Science and Letters U. Jondelius et al. • Nemertodermatida are basal bilaterians Fig. 1 continued © The Norwegian Academy of Science and Letters • Zoologica Scripta, 31, 2, April 2002, pp201 – 215 207 Nemertodermatida are basal bilaterians • U. Jondelius et al. followed by the Nemertodermatida. During our initial analyses, the sequence identified as oriIginating from Nemertinoides elongatus (GenBank Accession no. U70084), which was used as a representative of the Nemertodermatida in previous analyses (Carranza et al. 1997; Zrzavy et al. 1998; Littlewood et al. 1999), did not group with the three nemertodermatid species sequenced by us, but with rhabditophoran Platyhelminthes. Therefore it was excluded from further analyses. There is no explicit support for the monophyly of the Lophotrochozoa in the most-parsimonious tree. The cladogram does not conflict with the Lophotrochozoa hypothesis, but the group is not retrieved as a monophylum. A group consisting of the representatives of Nematomorpha, Gastrotricha, Rotifera, Gnathostomulida and Acanthocephala branches basally (between the nemertodermatids and the rest of the Bilateria) in the most-parsimonious tree. The position of the Gastrotricha, Gnathostomulida and Rotifera has been difficult to determine in other studies based on 18S rDNA (Giribet et al. 2000) and their basal position here should be viewed with caution. Maximum likelihood analyses The tree resulting from the maximum likelihood analysis of the 18S rDNA data set is shown in Fig. 2. The branching order of the Acoela and the Nemertodermatida, highly supported by four-cluster likelihood mapping (98 and 82%, respectively), is identical to that of the parsimony analysis. The deuterostome clade is the sister group of the Ecdysozoa and Lophotrochozoa, which are recovered as monophyletic groups. In this tree, the Nematomorpha, Rotifera (only the single species which passed the relative rate test was included) and Gastrotricha branch within the Ecdysozoa or the Lophotrochozoa. The Gnathostomulida and the Acantocephala did not pass the relative rate test and were not included in this analysis. The resolution within the protostome clade, which includes the flatworm groups Rhabditophora and Catenulida, is higher than in the parsimony analyses. The analysis of the 18S data without the outgroup gave a tree in which the Nemertodermatida emerge from the branch leading to the deuterostomes, whereas ecdysozoans and lophotrochozoans are grouped together ( Fig. 3). We also tested three alternative topologies (using the comparison allowed by the user tree option in FASTDNAML) by forcing the nemertodermatids at three different positions: (1) at the base of the Ecdysozoa, (2) at the base of the Lophotrocozoa, (3) at the base of the Platyhelminthes within the Lophotrochozoa. In the three cases, the likelihood score of the tree was significantly worse than the score of the tree shown in Fig. 2 (difference in LnL: –13.56, standard deviation = 5.39 for trees 1 and 2; –20.61, standard deviation = 6.69 for tree 3). Combined 18S rDNA and mitochondrial gene analyses Parsimony jackknifing ( Farris et al. 1996) of the concatenated 18S rDNA and mitochondrial nucleotide sequences supported a clade consisting of the Nemertodermatida and all other Bilateria, but excluding the Acoela (not shown). The maximum likelihood analysis (Fig. 4) retrieved a tree similar to the 18S rDNA maximum likelihood tree (Fig. 2), except that there is no support for the Ecdysozoa ( Nematoda and Arthropoda do not form a monophylum). The branch support for the Acoela and the Nemertodermatida as basal clades of the Bilateria was high (80 and 88%, respectively). Removal of the most variable positions in the maximum likelihood analyses still resulted in strong support for the basal position of acoels and nemertodermatida (92 and 82%). Moreover, we found our sequences of Nemertodermatida and Acoela to have the standard invertebrate mitochondrial genetic code, not the modification of the mitochondrial genetic code previously reported from the Rhabditophora ( Telford et al. 2000). The rhabditophoran Microstomum lineare did share the modifications of other Rhabditophora. Discussion The results presented here strongly support the hypothesis that Nemertodermatida is a clade of basal bilaterians, separate from the Acoela, and not closely related to the Rhabditophora. In addition, the basal position of acoels is further reinforced. Altogether, this renders the Platyhelminthes a non-monophyletic assemblage which, awaiting the precise position of the Catenulida, must be split into three separate monophyletic clades: Acoela, Nemertodermatida and Rhabditophora + Catenulida. The finding that the sequence of Nemertinoides elongatus used in all previous analyses did not group with the three nemertodermatid species sequenced here, strongly suggests that it is a sequencing artefact or originated from an undisclosed rhabditophoran flatworm. The position of Nemertodermatida and Acoela separate from Rhabditophora is further supported by the recent finding of a new molecular synapomorphy also corroborated in this work: acoels and nemertodermatids together with catenulids have the standard invertebrate mitochondrial genetic code and do not share the previously reported modifications of the mitochondrial genetic code in the Rhabditophora ( Telford et al. 2000). Fig. 2 Tree resulting from the maximum likelihood analysis of the 18S ribosomal DNA data set (FASTDNAML, jumble and global options). Branch support for the Acoela and the Nemertodermatida are indicated at the corresponding nodes (calculated with PUZZLE 4.0). The tree illustrates the position of the Nemertodermatida as the sister group to the rest of the Bilateria except the Acoela. The Rabditophora + Catenulida clade branches within the Lophotrochozoa. For species names and taxonomical assignment see Table 2. Scale bar indicates the number of substitutions per position. 208 Zoologica Scripta, 31, 2, April 2002, pp201– 215 • © The Norwegian Academy of Science and Letters U. Jondelius et al. • Nemertodermatida are basal bilaterians © The Norwegian Academy of Science and Letters • Zoologica Scripta, 31, 2, April 2002, pp201 – 215 209 Nemertodermatida are basal bilaterians • U. Jondelius et al. Fig. 3 Unrooted tree resulting from the analysis of 18S ribosomal DNA data of bilaterian phyla not including the Acoela ( FASTDNAML, jumble and global options). The three main bilaterian clades are highlighted. The Nemertodermatida emerge at the base of the deuterostomian branch (for more details, see text). For clarity, the names of the species are not included. Scale bar indicates the number of substitutions per position. To avoid theoretically possible ‘long branch’ problems, four different strategies were followed. In the parsimony analyses we (1) recoded the nucleotide data into triplets to reduce branch length and homoplasy; and (2) included a large number of terminals (104 taxa) to break up branches and avoid sampling artefacts. In the maximum likelihood analyses we (3) used species that passed the relative rate test in previous analyses (Ruiz-Trillo et al. 1999) together with two nemertodermatid species reported here, and (4) performed one analysis with diploblasts and acoels removed. These widely different approaches produced consistent results with respect to the position of the Acoela and the Nemertodermatida. Parsimony analysis of the triplet-coded data set yielded a tree where ecdysozoans and lophotrochozoans form a polytomy. The low resolution within the protostomes might have been anticipated because 210 recoding nucleotides into triplets reduces the number of characters and, therefore, the resolution. Some ‘pseudocoelomate’ clades (see Fig. 1) occupy positions basal to the Deuterostomia and Protostomia in the most-parsimonious tree. This position must be regarded as highly tentative. While there is no generally accepted position for the Gastrotricha, Gnathostomulida, Rotifera and Acanthocephala, morphological synapomorphies uniting the latter three taxa are known (Rieger & Tyler 1995; Sorensen et al. 2000). Most of the ‘pseudocoelomate’ representatives did not pass the relative rate test (see Table 2) and thus their position could not be reconstructed in the maximum likelihood analysis. The position of the single nematomorph Gordius aquaticus in the most-parsimonious cladogram is clearly at odds with the current mainstream view of the Nematomorpha as part of the Ecdysozoa. Inclusion of Zoologica Scripta, 31, 2, April 2002, pp201– 215 • © The Norwegian Academy of Science and Letters U. Jondelius et al. • Nemertodermatida are basal bilaterians Fig. 4 Tree resulting from the maximum likelihood analysis of a combined 18S ribosomal DNA and mitochondrial data set ( FASTDNAML, jumble and global options). Branch support for the Acoela and the Nemertodermatida is shown at the corresponding nodes. The Acoela and the Nemertodermatida are, as in most-parsimonious and maximum likelihood trees based on 18S ribosomal DNA data alone, the two basalmost branches of the Bilateria. For species names see Table 3. Scale bar indicates the number of substitutions per position. a larger taxonomic sample of Nematomorpha and Nematoda might have produced a different result. The Deuterostomia, Ecdysozoa and Lophotrochozoa are retrieved in the maximum likelihood trees based on taxa that passed the relative rate test. In these maximum likelihood trees (Fig. 2), Nemertodermatida, together with the Acoela, branch as the most basal groups. Furthermore, removing diploblasts and acoels still resulted in a basal position for the nemertodermatids (Fig. 3), which never grouped with the © The Norwegian Academy of Science and Letters • Zoologica Scripta, 31, 2, April 2002, pp201 – 215 Rhabditophora. In addition, nemertodermatids do not branch within any of the three bilaterian clades: Deuterostomia, Ecdysozoa and Lophotrochozoa. This is again an indication of their basal bilaterian position. Adding mitochondrial nucleotide sequences to the 18S rDNA sequences and analysing the concatenated sequences by parsimony jackknifing and maximum likelihood (Fig. 4) did not increase the resolution. The resulting maximum likelihood tree is similar to the maximum likelihood tree based on 18S rDNA sequences 211 Nemertodermatida are basal bilaterians • U. Jondelius et al. alone (Fig. 2), but the Ecdysozoa are not retrieved. Compared with the bootstrap values for the 18S rDNA maximum likelihood tree in Fig. 2, the value for the nemertodermatids increases (82 to 88%) while it decreases for the acoels (98 to 80%). This seems to indicate that mitochondrial sequences add some useful phylogenetic information for nemertodermatids but not for acoels. However, these changes in bootstrap values might also be due to the more restricted species sampling of these analyses (19 species compared with 104 in most-parsimonious and 66 in maximum likelihood analyses). We performed a maximum likelihood analysis of the mitochondrial data alone (COI and Cytb first and second positions) which supported the basal position of the Acoela separate from the Rhabditophora (77% support in PUZZLE), but the position of the Nemertodermatida was not supported. Altogether, the most-parsimonious and maximum likelihood analyses agree with regards the position of the Nemertodermatida. Both support a basal position for nemertodermatids, with very high support values in maximum likelihood trees. It is worth noting that the position of the Nemertodermatida and the Acoela as the two basalmost branches in the Bilateria is also retrieved when the data are analysed in nucleotide sequence form with parsimony analysis, i.e. without triplet coding. Thus, it appears that there were no LBA artefacts affecting their positions to begin with. The basal phylogenetic position for Acoela and Nemertodermatida separate from Rhabditophora is also supported from another set of data. First, acoel embryonic cleavage and cell lineage ( Henry et al. 2000) show them to exhibit duet spiral cleavage and endomesoderm as the sole source of mesoderm compared with the canonical quartet spiral cleavage and ecto- and endomesoderm in the Rhabditophora and most spiralian lophotrochozoans. An endodermal origin of mesoderm is considered ancestral. Moreover, duet cleavage is actually more bilateral than spiral cleavage (Henry et al. 2000), strongly suggesting that duet cleavage and typical quartet cleavage are not related. Finally, acoels and nemertodermatids do not possess protonephridia. In Ehler’s (1985) hypothesis, the lack of protonephridia in Nemertodermatida and Acoela is regarded as an apomorphy, deviating from the ground plan of the Platyhelminthes. However, under our phylogenetic hypothesis, the absence of protonephridia is a plesiomorphic state. The presence of protonephridia or homologues, on the other hand, is a synapomorphy uniting all Bilateria except acoels and nemertodermatids. Despite the basal positioning of both acoels and nemertodermatids, the clade Acoelomorpha (Acoela + Nemertodermatida; Ehlers 1985) was never retrieved in our analyses. Similarities in the structure of the epidermal cilia (interconnecting ciliary rootlets and capped cilia) and the lack of protonephridia are synapomorphies proposed for the Acoelomorpha. The phylogeny of the Nemertodermatida was 212 reconstructed by Lundin (2000) using morphological characters. His study was performed under the assumption of platyhelminth monophyly, and thus is not a critical test of our hypothesis: only taxa belonging to the Nemertodermatida, Acoela, Rhabditophora and the marine worm Xenoturbella were included. Although Lundin (2000) seems to prefer the Acoelomorpha hypothesis, his results are entirely compatible with the results presented here after adjusting for our much larger taxonomic sample. Under our hypothesis, the ciliary structures are interpreted as plesiomorphic characters present in the last common ancestor of the Bilateria or independently evolved. It should be noted that knowledge of detailed ciliary morphology of the Bilateria is scant; these features may be more common than assumed when the Acoelomorpha hypothesis was conceived. Recent studies of neuroanatomy also support separate phylogenetic positions for Acoela, Nemertodermatida and Rhabditophora and other bilaterians. A true brain with neuropile is absent in the former two groups (Raikova et al. 1998; Reuter et al. 1998). The nemertodermatids Nemertoderma westbladi and Meara stichopi have a very simple nervous system with weak or no centralization. The patterns of neurotransmitters (5-hydroxytryptamine (serotonin) and FMRfamide) in the two species are different from the two dissimilar patterns known from the Acoela and the Rhabditophora (Raikova et al. 2000). Finally, the Acoelomorpha hypothesis receives no support from sperm morphology. The Nemertodermatida have uniflagellate spermatozoa with the common 9 + 2 axonemal pattern, which probably evolved from sperm of Franzén’s ‘primitive type’ (Lundin & Hendelberg 1998). Acoel spermatozoa bear two flagella with reverse orientation and sometimes a modified axoneme structure (Raikova et al. 2001). Although the Bilateria are widely recognized as a monophyletic group, the characterization of the bilaterian ground pattern is actually far from simple. This is because the reconstruction of characters of the last common bilaterian ancestor requires knowledge of early bilaterian phylogeny. If a small and structurally simple acoel-like ancestor is hypothesized, the rest of the bilaterian phyla evolved by stages of increasing size and complexity. This widely held notion has recently been substituted, arguing that acoelomate and pseudocoelomate animals are secondarily simplified. Instead, based on a basal split of the Bilateria into two coelomate clades, a rather complex organism (the so-called ‘Urbilateria’) has been proposed as the ancestral bilaterian. Under this new view the bilaterian ancestor was large and possessed a mouth and anus, coelom, segments and, very likely, some sort of appendages (Rieger 1986; Kimmel 1996; De Robertis 1997; Holland et al. 1997; Adoutte et al. 1999, 2000). However, as pointed out by Jenner (2000), the provided phylogenies are heavily pruned, leaving out several ‘minor’ phyla, namely basal ecdysozoans and lophotrochozoans, and the proposed expression pattern Zoologica Scripta, 31, 2, April 2002, pp201– 215 • © The Norwegian Academy of Science and Letters U. Jondelius et al. • Nemertodermatida are basal bilaterians homologies are unreliable and exhibit a strong taxon selection. A second scenario suggests a small ciliated primary larva with a population of set-aside cells [reviewed in Peterson et al. (2000)] as the ancestral bilaterian. This scenario hinges on the assumption that ‘maximal indirect development’ is primitive for bilaterians, direct development being derived (Cameron et al. 1998; Peterson et al. 2000). This is, at the most, an untested assumption. Moreover, the difficulties of homologizing the so-called primary larvae in different groups suggest that set-aside cells are not homologous but convergent. In addition, the phylogenies provided are again highly pruned and biased towards coelomate, indirectly developing bilaterians. A final, radically different, scenario has suggested a complex, tailed and segmented ancestor, bearing a dorsal brain, serially repeated kidneys, gonocoels and a contractile blood vessel, thought to have evolved from colonial cnidarian-grade organisms (Dewel 2000). Although novel in conception, the evidence to back it up is scant and unconvincing. The new data summarized in our phylogenetic hypothesis necessitate completely different parsimony-based inferences about the lifecycle and other characteristics present in the last common ancestor of the extant Bilateria. The Acoela and the Nemertodermatida do not possess a true brain or protonephridia. If our hypothesis is accepted, these characters are morphological synapomorphies of the higher Bilateria [see also Haszprunar (1996)]. We propose the name Nephrozoa for Bilateria excluding the Nemertodermatida and the Acoela. Nemertodermatids and acoels are minute, unsegmented bottom dwelling worms with internal fertilization and direct development. Our results therefore imply that the last common bilaterian ancestor was small, benthic, without segments and coelomic cavities, and lacked a planktonic larval stage. The position of acoels and nemertodermatids at the base of bilaterians has two further implications at different phylogenetic levels. First, if they arose from a cnidarian-like diploblast ancestor, the planula-like features of the Acoela and Nemertodermatida suggest they may have arisen by progenesis (attainment of sexual maturity in larval forms) from a planula-like larva. This is at variance with the traditional planuloid–acoeloid hypothesis, according to which the gastrula-like planuloid ancestor was a fully mature adult, from which both cnidarians and all bilaterians, via an acoeltype organism, arose [reviewed in Willmer (1990)]. Second, if acoels and nemertodermatids are corroborated as basal to all other bilaterians, they could be instrumental in determining the character states of the ancestral Bilateria and so greatly further our understanding of the evolution of the higher bilaterians. Among such characters, the evolution and further deployment of the mesoderm and the nervous system, the embryological origin of the excretory system, and the transition from a single body axis to two orthogonal body axes, are worthy of exploration. © The Norwegian Academy of Science and Letters • Zoologica Scripta, 31, 2, April 2002, pp201 – 215 We regard the hypothesis presented here as a current best estimate of the phylogenetic position of the Nemertodermatida and Acoela. It will be tested through the acquisition and analysis of new molecular or morphological data on a Metazoa-wide basis. Acknowledgements We thank Drs J. S. F. Farris and M. Källersjö for their help with the parsimony triplet analysis and the staff at the Marine Biological Laboratories at Espegrend, Norway, and Kristineberg, Sweden, for their assistance with collecting the material. We also thank Dr J. Boore for the primers, Dr M. Reuter for sending specimens, and Professor R. M. 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