Filling a gap in the phylogeny of flatworms: relationships within the Rhabdocoela (Platyhelminthes), inferred from 18S ribosomal DNA sequences Blackwell Publishing Ltd WIM R. WILLEMS, ANDREAS WALLBERG, ULF JONDELIUS, DAVID T. J. LITTLEWOOD, THIERRY BACKELJAU, ERNEST R. SCHOCKAERT & TOM J. ARTOIS Accepted: 1 September 2005 doi:10.1111/j.1463-6409.2005.00216.x Willems, W. R., Wallberg A., Jondelius, U., Littlewood, D. T. J., Backeljau, T., Schockaert, E. R. & Artois, T. J. (2006). Filling a gap in the phylogeny of flatworms: relationships within the Rhabdocoela (Platyhelminthes), inferred from 18S ribosomal DNA sequences. — Zoologica Scripta, 35, 1–17. The phylogeny of the Rhabdocoela, a species-rich taxon of free-living flatworms, is reconstructed based on complete 18S rDNA sequences. The analysis includes 62 rhabdocoels and 102 representatives of all major flatworm taxa. In total, 46 new sequences are used, 41 of them from rhabdocoel species, five from proseriates. Phylogenetic analysis was performed using maximum parsimony and Bayesian inference. Clade support was evaluated with parsimony jackknifing, Bremer support indices and Bayesian posterior probabilities. The resulting cladogram corroborates that the Rhabdocoela is monophyletic, but its sister group remains uncertain. The ‘Dalyellioida’ and the ‘Typhloplanoida’, both former rhabdocoel subtaxa, are polyphyletic. Within the Rhabdocoela the monophyletic Kalyptorhynchia, characterized by a muscular proboscis, forms the sister group of all other rhabdocoels. The Schizorhynchia is a monophyletic subtaxon of the Kalyptorhynchia, with the split proboscis as a synapomorphy. Except for the Dalyelliidae and the Typhloplanidae, both freshwater taxa, none of the ‘families’ previously included in the ‘Typhloplanoida’ and the ‘Dalyellioida’ appears to be monophyletic. As a result of this analysis, three existing and four new taxon names are formally defined following the rules of the Phylocode. Wim R. Willems, Research Group Biodiversity, Phylogeny and Population Studies, Centre for Environmental Sciences, Hasselt University, Campus Diepenbeek, Agoralaan Building D, B-3590 Diepenbeek, Belgium. E-mail: wim.willems@uhasselt.be Andreas Wallberg, Systematic Zoology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, SE-752 36 Uppsala, Sweden. E-mail: andreas.wallberg@ebc.uu.se Ulf Jondelius, Systematic Zoology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, SE-752 36 Uppsala, Sweden. E-mail: ulf.jondelius@ebc.uu.se David T. J. Littlewood, Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK. E-mail: t.littlewood@nhm.ac.uk Thierry Backeljau, Department of Invertebrates, Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B-1000 Brussels, Belgium. E-mail: thierry.backeljau@naturalsciences.be Ernest R. Schockaert, Research Group Biodiversity, Phylogeny and Population Studies, Centre for Environmental Sciences, Hasselt University, Campus Diepenbeek, Agoralaan Building D, B-3590 Diepenbeek, Belgium. E-mail: ernest.schockaert@uhasselt.be Tom J. Artois, Research Group Biodiversity, Phylogeny and Population Studies, Centre for Environmental Sciences, Hasselt University, Campus Diepenbeek, Agoralaan Building D, B-3590 Diepenbeek, Belgium. E-mail: tom.artois@uhasselt.be Introduction The first comprehensive phylogenetic analysis of the Platyhelminthes using Hennigian argumentation was made by Ehlers (1985). This analysis was mainly based on morphological (including many ultrastructural) characters. It showed that the Turbellaria is paraphyletic, but that the Cestoda (Cercomeromorpha) and the Trematoda are monophyletic, together forming the monophyletic taxon Neodermata (with a neodermis). More recent studies, using molecular data and more elaborate cladistical techniques (e.g. Katayama et al. © The Norwegian Academy of Science and Letters 2005 • Zoologica Scripta, 35, 1, January 2006, pp1 – 17 1 18S rDNA phylogeny of Rhabdocoela • W. R. Willems et al. 1996; Carranza et al. 1997; Littlewood et al. 1999a,b; Joffe & Kornakova 2001; Littlewood & Olson 2001; Zamparo et al. 2001; Lockyer et al. 2003; see Baguñà & Riutort 2004 for a review) confirmed the monophyly of the Neodermata, but also showed that the relationships among the various other flatworm taxa are much more complex than was suggested by Ehlers (1985). Recent molecular data even suggest that the Platyhelminthes as a whole is polyphyletic, the Acoela and Nemertodermatida being basal bilaterians (Zrzavy et al. 1998; Ruiz-Trillo et al. 1999, 2002, 2004; Telford et al. 2000, 2003; Baguñà et al. 2001a; Jondelius et al. 2002; but see Tyler 2001 for an alternative view). Ehlers (1985) considered the Rhabdocoela as consisting of the ‘Typhloplanoida’, the ‘Dalyellioida’ and the parasitic Neodermata. However, since no apomorphies for either the ‘Typhloplanoida’ (including the Kalyptorhynchia) or the ‘Dalyellioida’ (including the Temnocephalida and other symbiotic taxa) could be put forward, both groups were regarded as nonmonophyletic and their exact position within the Rhabdocoela was left open. Analyses of rDNA sequences showed that the sister group of the Neodermata is included neither within the ‘Dalyellioida’ nor the ‘Typhloplanoida’, but probably consists of a large clade containing most of the neoophoran taxa (Littlewood et al. 1999a,b; Baguñà et al. 2001b; Joffe & Kornakova 2001; Littlewood & Olson 2001; Norén & Jondelius 2002; Lockyer et al. 2003; review in Baguñà & Riutort 2004). Therefore, the name Rhabdocoela is now commonly used by most authors for a group containing the ‘Dalyellioida’, ‘Typhloplanoida’, Kalyptorhynchia and Temnocephalida, the latter two being monophyletic. In fact, this is consistent with its most traditional use (and is a synonym of the name Neorhabdocoela of Meixner 1938). Currently, all recent molecular studies agree on the monophyly of the Rhabdocoela, although relationships within the taxon are still unclear. The main reason for this is probably poor taxon sampling. In phylogenetic analyses based on molecular data (18S rDNA) including Rhabdocoela, only a few rhabdocoel sequences were used: five by Lockyer et al. (2003), 12 by Littlewood et al. (1999a,b), Joffe & Kornakova (2001) and Littlewood & Olson (2001), and 20 by Norén & Jondelius (2002). The main results of these studies can be summarized as follows: (1) the Kalyptorhynchia, sometimes included within the ‘Typhloplanoida’ (see Ehlers 1985), is monophyletic and probably forms the sister group to all other rhabdocoels; (2) neither the ‘Typhloplanoida’ nor the ‘Dalyellioida’ are monophyletic; (3) there is some evidence for a freshwater clade containing the Typhloplanidae Graff, 1905 (‘Typhloplanoida’) and the Dalyelliidae Graff, 1905 (‘Dalyellioida’) and perhaps the Temnocephalida (‘Dalyellioida’) (see Joffe & Kornakova 2001; Watson 2001). The main objective of this study is to reveal the relationships within the Rhabdocoela by including a much larger number 2 of sequences than in any previous study. With these data we aimed at resolving the following questions: (1) What are the relationships of the various typhloplanoid ‘families’ within the Rhabdocoela? (2) What is the position of the Kalyptorhynchia? (3) Is there in fact a ‘freshwater clade’ consisting of members of the ‘Typhloplanoida’ and the ‘Dalyellioida’? Apart from the internal relationships, this study also set out to reveal possible sister group relations of the Rhabdocoela. Materials and methods Taxon sampling Forty-one specimens of 39 rhabdocoel species (24 ‘Typhloplanoida’ including Ciliopharyngiella constricta, 1 ‘Dalyellioida’ and 14 Kalyptorhynchia; see Table 1) were collected in both freshwater and marine habitats. Marine specimens were extracted from the sediment or from algae using the MgCl2 decantation method, while the freshwater specimens were collected by the oxygen depletion method (see Schockaert 1996). The specimen of Mesostoma thamagai Artois et al., 2004 was collected after inundating a sediment sample containing the resting propagules of this species from an ephemeral rock pool in Botswana (for details see Artois et al. 2004). The animals were starved for several hours up to one day to prevent DNA contamination by gut contents. Specimens were fixed in 96% ethanol and stored at 4 °C until DNA extraction. Five as yet undescribed species are included in the analysis, but to avoid creating nomina nuda, they are not given a species name here. Promesostoma sp. from New Caledonia clearly belongs to the taxon Promesostoma Graff, 1882 and its description has been submitted for publication. Gaziella sp. from the eastern Mediterranean is clearly identifiable as a species of Gaziella De Clerck and Schockaert, 1995. However, lack of material prevents its formal description. A third species, Castrada sp. from northern Sweden, could not be identified at the species level, but certainly belongs to the taxon Castrada Schmidt, 1861 (sensu Luther 1963). Two more species, ‘stradorhynchus terminalis’ and ‘arrawarria inexpectata’, belong to the kalyptorhynch taxon Polycystididae and are described in a submitted manuscript. Both species names are placed between quotation marks to stress that both are undescribed species. The sequence of ‘arrawarria inexpectata’ was already used by Littlewood et al. (1999b) and therein named Arrawarria gen. nov. Apart from the new rhabdocoel sequences, five proseriate species were sequenced (Table 1). Additional sequences (21 rhabdocoel and 97 nonrhabdocoel) were extracted from GenBank (Table 2), using BLAST search (Altschul et al. 1997). Species of which more than one sequence was available are numbered (e.g. Geocentrophora baltica 1, G. baltica 2). In total, 164 sequences are included in the analyses, 62 of them from 57 different rhabdocoel species. Paromalostomum fusculum, Zoologica Scripta, 35, 1, January 2006, pp1 – 17 • © The Norwegian Academy of Science and Letters 2005 W. R. Willems et al. • 18S rDNA phylogeny of Rhabdocoela Table 1 List of all new Proseriata and Rhabdocoela sequences included in this study, with their GenBank accession number and geographical origin (§: sequences provided by Littlewood and Webster). PROSERIATA RHABDOCOELA — Kalyptorhynchia RHABDOCOELA — ‘Typhloplanoida’ RHABDOCOELA — ‘Dalyellioida’ Species Accession No. Location Archimonocelis oostendensis Martens & Schockaert, 1981 Cirrifera sopottehlersae Noldt & Jouk, 1988 Coelogynopora axi Sopott, 1972 Pseudomonocelis ophiocephala (Schmidt, 1861) Meixner, 1943 Pseudomonocelis ophiocephala§ Acrorhynchides robustus Karling, 1931 Gyratrix hermaphroditus Ehrenberg, 1831 Karkinorhynchus bruneti Schilke, 1970 Mesorhynchus terminostylus Karling, 1956 Phonorhynchus helgolandicus (Metschnikow, 1863) Graff, 1905 Polycystis naegelii Kölliker, 1845 Proschizorhynchus triductibus Schilke, 1970 Schizochilus caecus L’Hardy, 1963 Schizochilus choriurus Boaden, 1963 Schizochilus marcusi Boaden, 1963 Schizorhynchoides caniculatus L’Hardy, 1963 ‘stradorhynchus terminalis’ Thylacorhynchus ambronensis Schilke, 1970 Zonorhynchus seminascatus Karling, 1956 Castrada lanceola (Braun, 1885) Luther, 1904 Castrada luteola Hofsten, 1907 Castrada viridis Volz, 1898 Castrada sp. Ciliopharyngiella constricta Martens & Schockaert, 1981 Ciliopharyngiella constricta§ Einarella argillophyla Luther, 1948 Einarella argillophyla§ Gaziella sp. Litucivis serpens Ax & Heller, 1970 Mesostoma lingua (Abildgaard, 1789) Schmidt, 1848 Mesostoma thamagae Artois et al., 2004 Olisthanella truncula (Schmidt, 1858) Luther, 1904 Phaenocora unipunctata Oersted, 1843 Promesostoma sp.§ Proxenetes flabellifer Jensen, 1878 Proxenetes puccinellicola Ax, 1960 Proxenetes quadrispinosus Den Hartog, 1966 Proxenetes simplex Luther, 1948 Proxenetes trigonus Ax, 1960 Ptychopera plebeia Beklemischev, 1927 Ptychopera westbladi Luther, 1943 Strongylostoma elongatum Hofsten, 1907 Styloplanella strongylostomoides Findenegg, 1924 Trigonostomum denhartogi (Karling, 1978) Willems et al., 2004 Trisaccopharynx westbladi Karling, 1940§ Castrella truncata (Abildgaard, 1789) Hofsten, 1907 AY775732 AY775733 AY775734 AY775735 AY775736 AY775737 AY775739 AY775740 AY775741 AY775742 AY775743 AY775744 AY775745 AY775746 AY775747 AY775748 AY775738 AY775749 AY775750 AY775751 AY775752 AY775753 AY775775 AY775754 AY775755 AY775756 AY775757 AY775776 AY775758 AY775759 AY775760 AY775761 AY775762 AY775763 AY775764 AY775765 AY775766 AY775767 AY775768 AY775769 AY775770 AY775771 AY775772 AY775773 AY775774 AY775777 Belgium, Oostende, Mariakerke Belgium, North Sea sandbank Germany, Sylt, Königshafen Greece, Thessaloniki, Perea Greece, Thessaloniki, Perea Germany, Sylt, Königshafen Sweden, Abisko Germany, Sylt, List Sweden, Kristineberg, Gullmaren Sweden, Kristineberg, Gullmaren Greece, Thessaloniki, Nea Michaniona Belgium, Zeebrugge Germany, Sylt, List Belgium, Oostende, Mariakerke Belgium, Knokke Germany, Sylt, List Australia, NSW, Coffs Harbour France, Wimereux Germany, Sylt, Königshafen Sweden, Abisko Sweden, Abisko Sweden, Abisko Sweden, Abisko Belgium, Oostende, Mariakerke Belgium, Oostende, Mariakerke Sweden, Kristineberg, Gullmaren Sweden, Kristineberg, Gullmaren Greece, Thessaloniki, Perea Germany, Sylt, List Sweden, Abisko Botswana, Thamaga Sweden, Abisko Belgium, Diepenbeek, De Maten New Caledonia, Nouméa, Ile Nou Belgium, Oostende Belgium, Knokke, Zwin Germany, Sylt, List Sweden, Kristineberg, Gullmaren Germany, Sylt, List Greece, Thessaloniki, Agia Trias Belgium, Knokke, Zwin Belgium, Diepenbeek Sweden, Abisko New Caledonia, Nouméa, Anse Vata Sweden, Kristineberg Sweden, Abisko Haplopharynx rostratus and five sequences of four species of Lecithoepitheliata (Geocentrophora sp., G. wagini, G. baltica 1, G. baltica 2 and G. sphyrocephala) were used as outgroups. DNA extraction, amplification and sequencing Genomic DNA was extracted from entire specimens using the DNeasy Tissue Kit (Qiagen) following the manufacturer’s protocol. The complete 18S rDNA gene, approximately 1800 bp long, was amplified using the primers TimA and TimB (see Table 3). Thermal cycling was started with an initial denaturation of 95 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 90 s with a final extension of 8 min at 72 °C. Using nested PCR (with the same cycling profile), the 1100 bp closest to the 5′ end of the 18S rDNA gene were amplified using TimA and 18S 1100R, while the © The Norwegian Academy of Science and Letters 2005 • Zoologica Scripta, 35, 1, January 2006, pp1 – 17 3 18S rDNA phylogeny of Rhabdocoela • W. R. Willems et al. Table 2 List of all additional flatworm species used in this study with their GenBank accession numbers and main references (*species for which a new sequence is also included, see Table 1). Numbered species names: different sequences of the same species extracted from GenBank. Species used to construct the probability model (see Materials and Methods: Alignment) are indicated with ‘M’. Species MACROSTOMIDA HAPLOPHARYNGIDA LECITHOEPITHELIATA PROSERIATA — Lithophora PROSERIATA — Unguiphora BOTHRIOPLANIDA ADIAPHANIDA — Fecampiidae ADIAPHANIDA — Genostomatidae ADIAPHANIDA — Urastomidae ADIAPHANIDA — Prolecithophora ADIAPHANIDA — Tricladida 4 In model Paromalostomum fusculum Ax, 1952 Haplopharynx rostratus Meixner, 1938 Geocentrophora baltica 1 (Kennel, 1883) Geocentrophora baltica 2 Geocentrophora sp. Geocentrophora sphyrocephala De Man, 1876 Geocentrophora wagini Timoshkin, 1984 Archiloa rivularis de Beauchamp, 1910 Archimonocelis crucifera Martens & Curini-Galletti, 1993 Archimonocelis staresoi Martens & Curini-Galletti, 1993 Archotoplana holotricha Ax, 1956 Calviria solaris Martens & Curini-Galletti, 1993 Coelogynopora gynocotyla Steinböck, 1924 Monocelis lineata (Müller, 1774) Oersted, 1844 Otoplana sp. Parotoplana renatae Ax, 1956 Nematoplana coelogynoporoides Meixner, 1938 Polystylophora novaehollandiae Curini-Galletti, 1998 Bothrioplana semperi Braun, 1881 Kronborgia isopodicola Blair & Williams, 1987 Ichthyophaga sp. Urastoma cyprinae 1 (Graff, 1882) Graff, 1903 Urastoma cyprinae 2 Urastoma sp. Allostoma neostiliferum Karling, 1993 Cylindrostoma fingalianum 1 (Claparède, 1861) Levinsen, 1878 Cylindrostoma fingalianum 2 Cylindrostoma gracilis Westblad, 1955 Euxinia baltica Meixner, 1938 Plagiostomum cinctum Meixner, 1938 Plagiostomum ochroleucum Graff, 1882 Plagiostomum striatum Westblad, 1956 Plagiostomum vittatum 1 (Frey & Leuckart, 1847) Jensen, 1883 Plagiostomum vittatum 2 Plicastoma cuticulata Brandtner, 1934 Protomonotresis centrophora Reisinger, 1924 Pseudostomum gracilis Westblad, 1955 Pseudostomum klostermanni (Graff, 1874) Graff, 1913 Pseudostomum quadrioculatum (Leuckart, 1847) Graff, 1911 Reisingeria hexaoculata Westblad, 1955 Scleraulophorus cephalatus Karling, 1940 Ulianinia mollissima Levinsen, 1879 Vorticeros ijimai Togawa, 1918 Artioposthia triangulata 1 (Dendy, 1895) Graff, 1896 Artioposthia triangulata 2 Artioposthia triangulata 3 Australoplana sanguinea (Moseley, 1877) Winsor, 1991 Australoplana sp. Baikalobia guttata (Gertsfeldt, 1858) Kenk, 1930 Bdelloura candida (Girard, 1850) Girard, 1852 Bipalium kewense Moseley, 1878 Bipalium sp. Bipalium trilineatum Stimpson, 1857 Caenoplana caerulea Moseley, 1877 Caenoplana sp. Crenobia alpina (Dana, 1766) Kenk, 1930 Cura pinguis (Weiss, 1909) Kenk, 1974 Dendrocoelopsis lactea Ichikawa & Okugawa, 1958 Dugesia iberica Gourbault & Benazzi, 1979 Accession No. Sequence reference M M M M M M M M AJ012531 AJ012511 AF167421 AF065417 U70079 D85089 AJ012509 U70077 AJ270151 AJ270152 AJ243676 AJ270153 AJ243679 U45961 D85090 AJ012517 AJ012516 AJ270161 AF051333 AJ012513 AJ012512 AF065428 AF167422 U70085 AF167420 AF065415 Littlewood et al. (1999a) Littlewood et al. (1999a) Jondelius et al. (2001) Norén & Jondelius (1999) Carranza et al. (1997) Katayama et al. (1996) Littlewood et al. (1999a) Carranza et al. (1997) Littlewood et al. (2000) Littlewood et al. (2000) Littlewood et al. (1999b) Littlewood et al. (2000) Littlewood et al. (1999b) Carranza et al. (1997) Katayama et al. (1996) Littlewood et al. (2000) Littlewood et al. (1999a) Littlewood et al. (2000) Baguñà et al. (2001b) Littlewood et al. (1999a) Littlewood et al. (1999a) Norén & Jondelius (1999) Jondelius et al. (2001) Carranza et al. (1997) Jondelius et al. (2001) Norén & Jondelius (1999) M M M M M M M AF051330 AF065416 AF167418 AF065418 AF065419 AF065420 AF051331 Baguñà et al. (2001b) Norén & Jondelius (1999) Jondelius et al. (2001) Norén & Jondelius (1999) Norén & Jondelius (1999) Norén & Jondelius (1999) Baguñà et al. (2001b) M M M M M M AF065421 AF065422 AF167419 AF065423 AF065424 AF065425 Norén & Jondelius (1999) Norén & Jondelius (1999) Jondelius et al. (2001) Norén & Jondelius (1999) Norén & Jondelius (1999) Norén & Jondelius (1999) M M M M M M M M M M M M M M M M M M M M AF065426 AF167423 AF065427 D85094 AF033038 AF033044 Z99945 AF033041 AF050434 Z99946 Z99947 AF033039 X91402 D85086 AF033040 AF048765 M58345 AF033043 D85087 M58343 Norén & Jondelius (1999) Jondelius et al. (2001) Norén & Jondelius (1999) Katayama et al. (1996) Carranza et al. (1998b) Carranza et al. (1998b) Carranza et al. (1998b) Carranza et al. (1998b) Carranza et al. (1998a) Carranza et al. (1998b) Carranza et al. (1998b) Carranza et al. (1998b) Mackey et al. (1996) Katayama et al. (1996) Carranza et al. (1998b) Carranza et al. (1998a) Riutort et al. (1992) Carranza et al. (1998a) Katayama et al. (1996) Riutort et al. (1992) M M M M M M M M M M M M M Zoologica Scripta, 35, 1, January 2006, pp1 – 17 • © The Norwegian Academy of Science and Letters 2005 W. R. Willems et al. • 18S rDNA phylogeny of Rhabdocoela Table 2 continued NEODERMATA RHABDOCOELA — ‘Dalyellioida’ RHABDOCOELA — ‘Typhloplanoida’ RHABDOCOELA — Kalyptorhynchia RHABDOCOELA — Temnocephalida Species In model Accession No. Sequence reference Dugesia japonica 1 Ichikawa & Kawakatsu, 1964 Dugesia japonica 2 Dugesia ryukyuensis Kwakatsu, 1976 Dugesia subtentaculata (Draparnaud, 1801) De Vries, 1986 Ectoplana limuli (Ijima & Kaburaki, 1916) Kaburaki, 1917 Girardia tigrina 1 (Girard, 1850) Girardia tigrina 2 Microplana nana Mateos, Giribet & Carranza, 1998 Microplana scharffi (Graff, 1896) Neppia montana (Nurse, 1950) Ball, 1974 Newzealandia sp. Phagocata sibirica (Sabussow, 1903) Kenk, 1974 Phagocata sp. Phagocata ullala Sluys, Ribas & Baguñà, 1995 Platydemus manokwari Beauchamp, 1962 Polycelis nigra (Müller, 1774) Polycelis tenuis Ijima, 1884 Procerodes littoralis (Ström, 1768) Hallez, 1893 Schmidtea mediterranea 1 (Benazzi et al., 1975) Schmidtea mediterranea 2 Schmidtea mediterranea 3 Schmidtea polychroa 1 (Schmidt, 1861) Schmidtea polychroa 2 Romankenkius libidinosus Sluys & Rohde, 1991 Uteriporus sp. Aspidogaster conchicola Baer, 1827 Caryophyllaeides ergensi Scholz, 1990 Dasyrhynchus pillersi Southwell, 1929 Diphyllobothrium stemmacephalum Cobbold, 1858 Echinococcus granulosus (Batsch, 1786) Rudolphi, 1805 Echinostoma caproni Richard, 1964 Fasciola gigantica Cobbold, 1856 Fasciola hepatica Linnaeus, 1758 Gyrodactylus rhodei Zitnan, 1964 Multicotyle purvisi Dawes, 1941 Phyllobothrium lactuca Van Beneden, 1850 Troglocephalus rhinobatidis Young, 1967 M M M M AF013153 D83382 AF050433 AF013155 Carranza et al. (1998a) Katayama et al. (1996) Carranza et al. (1998a) Carranza et al. (1998b, 1999) M M M M M M M M M M M M M M M M M M M M M D85088 AF013156 AF013157 AF033042 AF050435 AF050432 AF050431 Z99948 AF013150 AF013149 AF048766 AF013151 Z99949 Z99950 M58344 U31084 U31085 AF013152 AF013154 Z99951 AF013148 AJ287478 AF286979 AJ287496 AF124459 U27015 L06567 AJ011942 AJ004969 AJ567670 AJ228785 AF286999 AJ228795 Katayama et al. (1996) Carranza et al. (1998b, 1999) Carranza et al. (1998b, 1999) Carranza et al. (1998b) Carranza et al. (1998a) Carranza et al. (1998a) Carranza et al. (1998a) Carranza et al. (1998b) Carranza et al. (1998a, 1999) Carranza et al. (1998b, 1999) Carranza et al. (1998a) Carranza et al. (1998a, 1999) Carranza et al. (1998b) Carranza et al. (1998b) Riutort et al. (1992, 1993) Carranza et al. (1996) Carranza et al. (1996) Carranza et al. (1998b, 1999) Carranza et al. (1998b, 1999) Littlewood et al. (1999a) Carranza et al. (1998b, 1999) Cribb et al. (2001); Littlewood & Olson (2001) Olson et al. (2001) Littlewood & Olson (2001) Olson & Caira (1999) Picon et al. (1996) Blair & Barker (1993) Littlewood (1999) Fernandez et al. (1998) Matejusova et al. (2003) Littlewood et al. (1999a) Olson et al. (2001) Littlewood et al. (1998); Littlewood & Olson (2001) Littlewood et al. (1999a) Jondelius et al. (2001) Littlewood et al. (1999a) Littlewood et al. (1999a) Norén & Jondelius (2002) Norén & Jondelius (2002) Littlewood et al. (1999a) Norén & Jondelius (2002) Turbeville et al. (1992); Riutort et al. (1992, 1993) Katayama et al. (1996) Norén & Jondelius (2002) Littlewood et al. (1999a) Carranza et al. (1997) Littlewood et al. (1999b) Norén & Jondelius (2002) Littlewood et al. (1999b) Littlewood et al. (1999a) Littlewood et al. (1999a) Littlewood et al. (1999a) Norén & Jondelius (2002) Littlewood et al. (1999a) Baguñà et al. (2001a) Udonella caligorum Johnston, 1835 Anoplodium stichopi Bock, 1925 Graffilla buccinicola Jameson, 1897 Microdalyellia rossi (Graff, 1911) Gieysztor, 1938 Provortex balticus (Schultze, 1851) Graff, 1882 Provortex tubiferus Luther, 1948 Pterastericola australis Cannon, 1986 Astrotorhynchus bifidus (McIntosh, 1874) Graff, 1905 Bothromesostoma personatum (Schmidt, 1848) Fuhrmann, 1894 Bothromesostoma sp. Maehrenthalia agilis (Levinsen, 1879) Graff, 1905 Mariplanella frisia Ax & Heller, 1970 Mesoscastrada sp. Mesostoma lingua* Trigonostomum penicillatum Schmidt, 1857 ‘arrawarria inexpectata’ Cheliplana cf. orthocirra Diascorhynchus rubrus Boaden, 1963 Gyratrix hermaphroditus* Phonorhynchus helgolandicus* Temnocephala sp. 1 Temnocephala sp. 2 © The Norwegian Academy of Science and Letters 2005 • Zoologica Scripta, 35, 1, January 2006, pp1 – 17 M M M M M M M M M M M M M M M AJ228796 AF167424 AJ012521 AJ012515 AJ312268 AJ312269 AJ012518 AJ312270 M58347 D85098 AJ312273 AJ012514 U70081 AJ243682 AJ312275 AJ243677 AJ012507 AJ012508 AJ012510 AJ312274 AJ012520 AF051332 5 18S rDNA phylogeny of Rhabdocoela • W. R. Willems et al. Table 3 Primers used in PCR and sequencing reactions. Primer Used in Primer sequence Reference Tim A Tim B 600F 1100R 18S4FB 18S4FBK 18S5F 18S5FK 18S7F 18S7FK PCR/Sequencing PCR/Sequencing PCR PCR Sequencing Sequencing Sequencing Sequencing Sequencing Sequencing 5′-AMCTGGTTGATCCTGCCAG-3′ 5′-TGATCCATCTGCAGGTTCACCT-3′ 5′-GGTGCCAGCAGCCGCGGT-3′ 5′-GATCGTCTTCGAACCTCTG-3′ 5′-CCAGCAGCCGCGGTAATTCCAG-3′ 5′-CTGGAATTACCGCGGCTGCTGG-3′ 5′-GCGAAAGCATTTRYCHAGDA-3′ 5′-THCTDGRYAAATGCTTTCGC-3′ 5′-GCAATAACAGGTCTGTGATGC-3′ 5′-GCATCACAGACCTGTTATTGC-3′ Norén & Jondelius (1999) Norén & Jondelius (1999) Modified after Norén & Jondelius (1999) Norén & Jondelius (1999) Norén & Jondelius (1999) Norén & Jondelius (1999) Modified after Norén & Jondelius (1999) Modified after Norén & Jondelius (1999) Norén & Jondelius (1999) Norén & Jondelius (1999) 1200 bp closest to the 3′ end were amplified with the primers 18S 600F and TimB, which gave approximately a 500 bp overlap. All PCR reactions were performed in 25 µL, using Promega PCR Core System I. These reactions, containing 0.2 µM of the respective forward and reverse primer, 1 mM of each dNTP, one-tenth volume of Taq DNA Polymerase buffer 10X, 1.5 mM MgCl2 and 1.25 U of Taq DNA Polymerase, were carried out on an Eppendorf Mastercycler Gradient. The PCR results (5 µL) were verified on a 1% agarose gel, stained with ethidium bromide. PCR products were purified with the Qiaquick PCR Purification Kit from Qiagen and stored at 4 °C. Sequencing was performed by the Genetic Service Facility of VIB (Flanders Interuniversity Institute for Biotechnology), using TimA, TimB and six internal primers (see Table 3) on an ABI 3730 DNA Analyser (Applied Biosystems) with the ABI PRISM BigDye Terminator cycle sequencing kit. The sequences were verified by forward and reverse comparisons using Chromas v. 1.45 (freeware from http:// www.technelysium.com.au/index.html). Three rhabdocoel species for which 18S rDNA sequences are available in GenBank (Gyratrix hermaphroditus, Mesostoma lingua and Phonorhynchus helgolandicus; see asterisked entries in Table 2) are also represented by a new sequence as an additional check on the sequence’s quality. For some of the specimens, both PCR reactions and sequencing were performed in DTJL’s laboratory by Bonnie Webster (§ in Table 1) using protocols outlined in Littlewood et al. (2000). All new sequences have been deposited in GenBank (http:// www.ncbi.nlm.nih.gov). Their accession numbers can be found in Table 1. Alignment A prior alignment of 94 sequences of rhabditophoran flatworms (M column in Table 2) based on secondary structures was downloaded from the SSU rRNA database (http:// www.psb.ugent.be/rRNA; see also Wuyts et al. 2004). This initial alignment was used to create a hidden Markov model 6 profile with the hmmbuild option in HMMER 2.3.2 (http://hmmer.wustl.edu/; see also Eddy 1998). This model contains probability parameters, which are estimated from the observed frequencies of residues and transitions in the initial multiple sequence alignment. With this probability model, HMMER was used to create an alignment (hmmalign option) of the new sequences (see Table 1) and 24 additional sequences from GenBank, mainly Neodermata sequences (see Table 2). This resulted in an alignment of 3522 base positions, which was edited using MacClade 4.06 (Maddison & Maddison 2003). In total, 1729 positions of the alignment were deleted because they had a gap in all except for one or two of the species. These positions would not have influenced the parsimony analysis, but would have required a larger amount of computational effort in the Bayesian analyses. Longer blocks of deleted nucleotides only appeared in the neodermatan species. Phylogenetic analyses The final data matrix consists of 1793 unambiguously alignable base positions for 164 species and was analysed using maximum parsimony and Bayesian inference. Prior to the analyses, base composition (% GC content) was calculated using PAUP* 4.0b10 (Swofford 2003) to account for possible base compositional bias. Parsimony analysis (with gaps treated as missing data) was performed using both PAUP* and TNT 1.0 (Goloboff et al. 2001), the former in combination with PAUPRat (Sikes & Lewis 2001). PAUPRat implements the parsimony ratchet (Nixon 1999), making the tree search more efficient. In PAUP* the Rat-search was performed once and the settings were as follows: nchar = 1793, random seed = 0, nreps = 200, pct = 15 (default; ideally between 5 and 25%, see Nixon 1999), wtmode = uniform, terse. For all the heuristic searches performed by PAUPRat, the default settings were used. In TNT the Rat-search was repeated 20 times (as recommended in the PAUPRat manual: Sikes & Lewis 2001), each with 500 iterations and about the same deletion frequency as above. Zoologica Scripta, 35, 1, January 2006, pp1 – 17 • © The Norwegian Academy of Science and Letters 2005 W. R. Willems et al. • 18S rDNA phylogeny of Rhabdocoela We employed two approaches to control for rate heterogeneity effects that may affect the results of a parsimony analysis: taxa that had the largest pairwise distances, and thus may constitute long branches, were removed from the dataset and a separate parsimony analysis was run. In addition, our Bayesian analysis was designed to take unequal rates into account. Bayesian inference (Rannala & Yang 1996; Mau & Newton 1997; Yang & Rannala 1997; Mau et al. 1999; Larget & Simon 1999) was performed in MrBayes 3.0b4 (Huelsenbeck & Ronquist 2001) under the general time-reversible model (GTR; Rodríguez et al. 1990), with discrete gamma-distributed rate variation among sites (Yang 1993, 1994) with four categories, and allowing for invariant sites (Gu et al. 1995; Waddell & Steel 1997). This model was chosen by MODELTEST 3.06 (Posada & Crandall 1998) as the model of DNA evolution that best fitted the data. Five independent runs were performed, each with 2 million generations and four chains (default temperature), sampled every 100 generations. Branch lengths were saved. An additional run with 10 million generations was also done to ensure that the analysis was running long enough to converge on a stable LnL value. Different parameters can converge at different rates (Huelsenbeck et al. 2002). Therefore both log-likelihood and tree length values were plotted against the generation number. The burn-in value was chosen in function of both parameters converging on a stable value. After discarding the trees sampled during the burn-in, the results were summarized in 95% majority rule consensus trees. All analyses were run on two 86 AMD 2800+ CPUs. The occurrence of long-branch attraction (see Felsenstein 1978) was additionally tested by omitting the four taxa with the largest mean pairwise distance compared to all other taxa from the parsimony analysis in PAUP*: Graffilla buccinicola Jameson, 1897, Plagiostomum ochroleucum Graff, 1882, Udonella caligorum Johnston, 1835 and Vorticeros ijimai Graff, 1899. Clade support Clade support was assessed by calculating jackknife values (Lanyon 1985; Siddall 1995), Bremer support values (Bremer 1988, 1994) and Bayesian posterior probabilities (see above for references). Bremer support (Bremer 1988, 1994) was calculated using TreeRot v2 (Sorenson 1999). This program generates a command file for PAUP*, which consists of the constraint statement for each node and the commands to search for the shortest tree incompatible with this node. The constrained searches to determine the Bremer support indices were done using the parsimony ratchet. For estimating nodal support in the parsimony analysis, character jackknifing was preferred to bootstrapping for its computational efficiency (see Farris 1998), and was performed with Xac (Farris 1997); character deletion frequency e−1, 1000 replicates, 3 random additions and branch swapping enabled. For Bayesian analysis nodal support was estimated by determining posterior probabilities with MrBayes. Monophyly of the Typhloplanoida and Typhloplanoida sensu lato (Typhloplanoida + Kalyptorhynchia; see Ehlers 1985) was tested by constructing two constraint trees in MacClade, one with a monophyletic Typhloplanoida, the other with a monophyletic Typhloplanida. A PAUPRat search in PAUP* with 200 iterations was run and Templeton (Templeton 1983) and Winning sites (Prager & Wilson 1988) tests were performed with PAUP* to determine whether these constraint solutions were significantly different from the most parsimonious solution. The constraint trees were also used to filter the trees sampled in the Bayesian analysis. Results Sequence data Within the alignment the length of the 18S rDNA fragment varied between 1308 bp (Dugesia iberica) and 1792 bp (Archimonocelis crucifera, Cirrifera sopottehlersae and Coelogynopora axi ). The GC content varied between 40.4% (Dugesia mediterranea 3) and 51.7% (Echinococcus granulosus) with an average of 45.3%. Remarkably, all neodermatans have a high GC content, ranging from 47.1% to 51.7%, whereas most of the triclads have a rather low value ranging between 40.7% and 47.4%. Representatives of the Rhabdocoela had a GC content which is scattered over almost the whole range, from 42.3% in Astrotorhynchus bifidus to 48% in Olisthanella truncula. The difference between these extremes (within the Rhabdocoela) was lower than the 8–10% value, which is often assumed to be the maximum value at which a biasing effect of compositional heterogeneity can occur (e.g. Galtier & Gouy 1995). Therefore, it is unlikely that GC bias consitutes a problem in our data set. Parsimony analysis Of the 1793 unambiguously alignable base positions in the final alignment, 468 sites were constant and 245 were parsimonyuninformative, resulting in 1080 parsimony-informative characters. The analysis in PAUP* combined with PAUPRat generated seven topologically distinct MP trees (length = 14 822 steps; CI = 0.180; RI = 0.678; RC = 0.122). The strict consensus of these trees is depicted in Figs 1–2. TNT ratchet analyses yielded trees of the same length and topology as PAUPRat. The analysis in which the taxa with the largest mean pairwise distance were excluded resulted in a tree of 14 022 steps with the same overall topology as that of the tree depicted in Figs 1–2. Bayesian inference Four out of five independent runs of 2 million generations converged after 110 000–250 000 generations, although with © The Norwegian Academy of Science and Letters 2005 • Zoologica Scripta, 35, 1, January 2006, pp1 – 17 7 18S rDNA phylogeny of Rhabdocoela • W. R. Willems et al. Fig. 1 Strict consensus of seven most parsimonious trees of 14 822 steps (CI = 0.180; RI = 0.678; RC = 0.122) obtained in PAUP* combined with PAUPRat (200 iterations). Bremer support values are indicated above each clade, jackknife values beneath. Clades with a posterior probability of ≤ 95% in the Bayesian analysis are indicated with dashed lines. Present taxonomic positions are indicated on the right. (§ and *: see Tables 1 and 2). Adiaphanida (A) is depicted in Fig. 2. 8 Zoologica Scripta, 35, 1, January 2006, pp1 – 17 • © The Norwegian Academy of Science and Letters 2005 W. R. Willems et al. • 18S rDNA phylogeny of Rhabdocoela Fig. 2 Adiaphanida. Part of the tree in Fig. 1, therein abbreviated as (A). Table 4 Burn-in (with respect to log-likelihood and tree length values) and mean log-likelihood values of six independent MrBayes runs. Run III failed to converge: LnL plot showed plateaus after 150 000, 850 000, 1 060 000 and 1 400 000 generations; TL plot after 250 000, 870 000, 1 030 000 and 1 370 000 generations (I–V: 2 M generations, VI: 10 M generations; burn-in value used in computing consensus tree is indicated in bold). Run Burn-in (LnL) Burn-in (TL) LnL I II III IV V VI 110 000 150 000 1 400 000 240 000 140 000 100 000 250 000 150 000 1 370 000 200 000 165 000 150 000 −67346.772 −67347.648 / −67343.042 −67344.184 −67345.293 different values for the burn-in according to log-likelihood and treelength values (see Table 4). The third run (no. III, see Table 4) did not converge, but the majority rule consensus tree (burn-in chosen at 250 000) was identical to the strict consensus of the MP trees. The 95% majority rule consensus trees were identical to the strict consensus of the parsimonious trees, depicted in Figs 1–2. Tree topology and clade support The tree in Figs 1–2 shows the results of both the parsimony and the Bayesian analysis. This tree is simplified in Fig. 3 by collapsing clades with jackknife ≤ 90% and by considering only the major taxa. The sister group relations of the major (ingroup) neoophoran taxa are far from resolved, which is evident from the tree in Figs 1–2 and 3. They form a large polytomy in a poorly supported clade (Bremer support 7; jackknife 75%; Bayesian posterior probability 100%), formed by the Rhadocoela, Lithophora, Unguiphora, Neodermata and Adiaphanida (= Prolecithophora + Tricladida + ‘turbellarian Revertospermatida’) and two isolated species, Ciliopharyngiella constricta and Bothrioplana semperi. It is beyond the scope of this contribution to discuss any of the other supported clades within these taxa. There is strong support for a monophyletic Rhabdocoela (BS 23; jackknife 98%; BPP 100%). Its sister group, however, cannot be indicated as yet. © The Norwegian Academy of Science and Letters 2005 • Zoologica Scripta, 35, 1, January 2006, pp1 – 17 9 18S rDNA phylogeny of Rhabdocoela • W. R. Willems et al. Fig. 3 Summary of results based on strict consensus of seven most parsimonious trees with the major taxa under consideration. Only clades with jackknife values of > 90% are indicated. One rectangle represents > 90% jackknife support, two represent > 95% and three, 100%; * clade with 75% support. Number of sequences used is indicated in parentheses following taxon names. Figure 4 shows the relationships within the Rhabdocoela. All clades except two (see Fig. 4: *) have jackknife support of 99–100% and all collapsed clades have jackknife support of ≤ 65%. The Rhabdocoela shows a trichotomy formed by Mariplanella frisia, the monophyletic Kalyptorhynchia and a clade containing all ‘Dalyellioida’ and all ‘Typhloplanoida’ (excl. Mariplanella frisia and Ciliopharyngiella constricta), hence referred to as Dalytyphloplanida (see Nomenclatural implications). There is support for a clade formed by the Dalytyphloplanida and M. frisia (BS 10; jackknife 81%; BPP 99% — see Figs 1–2). Within the Kalyptorhynchia there is strong support for a monophyletic Schizorhynchia, but not for a monophyletic Eukalyptorhynchia. None of the ‘families’ within the Schizorhynchia nor the Eukalyptorhynchia can be recognized. Within the Dalytyphloplanida there are two highly supported clades, the Neodalyellida and the Neotyphloplanida (see Nomenclatural implications). In the Neodalyellida we find all marine dalyellioids (including the symbionts) and two marine typhloplanoids: Trisaccopharynx westbladi (Solenopharyngidae) and Einarella argillophyla (Promesostomidae). The Neotyphloplanida consists of a polytomy with Gaziella sp. and four clades: the (freshwater) Temnocephalida Blanchard, 1849, the (freshwater) Dalyelliidae Graff, 1905, the 10 (freshwater) Typhloplanidae Graff, 1905, and a clade with all marine typhloplanoids (+ the freshwater Styloplanella strongylostomoides, so far included in the Typhloplanidae). This last taxon, with all marine typhloplanoids, receives the name Thalassotyphloplanida (see Nomenclatural implications). There is evidence for the monophyly of the Dalyelliidae and the Typhloplanidae but not for other taxa of the family level. The Templeton and Winning sites tests both found the 14 distinct MP trees to be significantly different at the 95% confidence level from the ‘Typhloplanoida’ and ‘Typhloplanoida’ s.l. (= Typhloplanoida + Kalyptorhynchia) constraint trees (P < 0.0001). Moreover, filtering all trees resulting from the Bayesian analyses, including the trees from run III (excluding 240 000 generations as burn-in), did not resolve any tree compatible with a monophyletic Typhloplanoida and Typhloplanoida s.l. Thus all our tests rejected the monophyly of Typhloplanoida s.l. and Typhloplanoida. In Fig. 5 all three measures of clade support are compared. Clades with jackknife and BPP values < 50% are not included in the graphs. From Fig. 5A, it appears that for clades with Bremer support values higher than > 10, the jackknife support was > 80%. The posterior probabilities were exceptionally high compared to the jackknife (Fig. 5B) and the Bremer support (Fig. 5C) values. These results confirm that BPP values may be misleadingly high, even with low jackknife support (see also Simmons et al. 2004). Discussion The reconstruction of the 18S rDNA gene tree for the taxa under consideration seems to be robust. The three different support values calculated here (Bremer support, jackknife and posterior probabilities) agreed in most cases (see Fig. 5). However, based on simulation studies, Bayesian posterior probabilities are said to be misleadingly high in comparison with jackknife values (Simmons et al. 2004) and bootstrap values (Suzuki et al. 2002; Alfaro et al. 2003; Douady et al. 2003; Erixon et al. 2003). From Fig. 5C, it is clear that in our empirical study Bayesian posterior probabilities are indeed considerably higher than the jackknife values. However, in the preferred tree (see Figs 1–2) clades with high jackknife values (> 90%) in most cases also have a high Bremer support value (> 10) and a very high posterior probability (> 95% and even 100% in most cases). Most of the deeper branches, i.e. the relationships between the major neoophoran taxa, are only weakly supported (see Figs 1–3), which could be an indication that the relationships between these taxa cannot be properly revealed with 18S rDNA alone. It highlights the necessity of searching for other molecular markers to sort out the relationships within the Platyhelminthes. In contrast, lower level clades within the Rhabdocoela (and within the other taxa) are strongly supported. The support values for the Rhabdocoela and well Zoologica Scripta, 35, 1, January 2006, pp1 – 17 • © The Norwegian Academy of Science and Letters 2005 W. R. Willems et al. • 18S rDNA phylogeny of Rhabdocoela Fig. 4 Rhabdocoela. Strict consensus of seven most parsimonious trees obtained in PAUP* combined with PAUPRat. Clades with jackknife values of < 90% are collapsed. Named (and unnamed) monophyletic taxa are on the right. The number of species in a terminal taxon is indicated in parentheses. Freshwater species are in bold, ‘Dalyellioida’ are underlined. All collapsed clades have < 65%, jackknife support, most remaining clades have 99–100%; those indicated with an asterisk have 94% support. supported clades within this taxon are given in Table 5, together with possible morphological apomorphies (see below). Another indication of the robustness of the tree is the congruence between the MP tree and the Bayesian tree. As Bayesian inference accounts for rate heterogeneity across sites (if the used model is corrected for this), the congruence between both methods indicates absence of long-branch attraction artifacts. This view is further supported by the fact that the same overall tree topology is found when taxa with the largest main pairwise distances in comparison to the other taxa are excluded from the parsimony analysis. Robustness of the rhabdocoelan part of the tree was further tested by enforcing the monophyly of the ‘Typhloplanoida’ and a monophyletic ‘Typhloplanoida + Kalyptorhynchia’. The results of the tests clearly showed that both groups are not monophyletic. At present, no molecular phylogenetic study deals excusively with the Rhabdocoela, which nevertheless is one of the largest taxa of free-living flatworms. Earlier molecular analyses that included more than one rhabdocoel sequence (e.g. Littlewood et al. 1999a,b; Baguñà et al. 2001b; Joffe & Kornakova 2001; Littlewood & Olson 2001; Norén & Jondelius © The Norwegian Academy of Science and Letters 2005 • Zoologica Scripta, 35, 1, January 2006, pp1 – 17 11 18S rDNA phylogeny of Rhabdocoela • W. R. Willems et al. Fig. 5 A–C. Comparison of Bremer support, jackknife and posterior probability (PP) values. Correlation between jackknife and Bremer support values (A), jackknife values and posterior probabilities (B), and Bremer support values and posterior probabilities (C). 2002; Lockyer et al. 2003) paid little attention to the relationships within the Rhabdocoela. All these studies are based on 18S rDNA, except for those by Littlewood et al. (1999b), Norén & Jondelius (2002) and Lockyer et al. (2003), which also include data on 28S rDNA. All the above studies report a monophyletic Rhabdocoela, albeit with varying nodal support (bootstrap values of < 50 – 77% in Littlewood et al. 1999a,b; Baguñà et al. 2001b; Joffe & Kornakova 2001; Littlewood & Olson 2001; jackknife value of 98–99% in the present study and in Norén & Jondelius 2002). With the present results the sister group of the Rhabdocoela cannot be identified as it forms part of a polytomy (see Fig. 3). Former studies found some support for a sister group relationship of the Rhabdocoela with the Adiaphanida (Littlewood et al. 1999a,b; Baguñà et al. 2001b; Joffe & Kornakova 2001; Littlewood & Olson 2001; Norén & Jondelius 2002; Lockyer et al. 2003). However, with bootstrap or jackknife values around 50%, none of these studies is convincing and with poorly supported clades collapsed, all cladograms would show the same polytomy as in our study. In our study, one of the members of the polytomy is Ciliopharyngiella constricta Martens & Schockaert, 1981. The taxon Ciliopharyngiella Ax, 1952 was formerly placed within the ‘Typhloplanoida’ (see Ehlers 1972), but its taxonomic position has been heavily debated based on morphological (see Ax 1952; Ehlers 1972) and ultrastructural data (see Brüggeman 1985; Sopott-Ehlers 1997, 1999, 2001) and now it appears that molecular data do not completely solve the problem either. Of all above mentioned studies, the one of Norén & Jondelius (2002), which includes 20 rhabdocoel species, has the most extensive taxonomic sampling within the Rhabdocoela, whereas the others include five (Lockyer et al. 2003) or 12 (Littlewood et al. 1999a,b; Joffe & Kornakova 2001; Littlewood & Olson 2001) species. Therefore the results of our analyses, which include 62 sequences of 57 different rhabdocoel species currently included within the Rhabdocoela, are best compared with those of Norén & Jondelius (2002). The tree topology within the Rhabdocoela is identical and Table 5 Overview of support values and possible morphological apomorphies for the Rhabdocoela and newly defined clades within this taxon. Jackknife (%) Bremer support BPP (%) Possible apomorphies 98 23 100 Kalyptorhynchia Schizorhynchia Dalytyphloplanida 100 100 99 31 16 21 100 100 100 Neodalyellida Neotyphloplanida Thalassotyphloplanida 99 100 100 12 29 19 100 100 100 pharynx bulbosus (?); terminal cell of protonephidia with single row of ribs (?); type C protonephridia (?); dense heel in sperm (?) proboscis; incorporation of axonemes in sperm split proboscis; loss of one axoneme in sperm presence of small dense granules, an axonemal spur, a group of longitudinal microtubules in the sperm and a fine connection between nuclear and plasma membranes (?) none none none Rhabdocoela 12 Zoologica Scripta, 35, 1, January 2006, pp1 – 17 • © The Norwegian Academy of Science and Letters 2005 W. R. Willems et al. • 18S rDNA phylogeny of Rhabdocoela clades that coincide with our Dalytyphloplanida, as well as with our Neodalyellida and Neotyphloplanida are apparent (see Norén & Jondelius 2002: Fig. 1). Previous morphological analyses dealing with the Rhabdocoela ( Jondelius & Thollesson 1993; Zamparo et al. 2001) are difficult to compare with, since the taxonomic composition is very different and because they include only a small number of terminals, almost all of them of the ‘family’-level. Nevertheless, it is possible to indicate morphological apomorphies for some of the rhabdocoelan clades (see Table 5). However, a large number of morphological aspects concern ultrastructural data, which should be interpreted with caution, as few taxa have been sampled and therefore these data are highly fragmentary. Good reviews of current knowledge on the ultrastructure of sperm and protonephridia can be found in Watson (2001) and Rohde (2001), respectively. Several contributions on the ultrastructure of other organs (e.g. male and female atrial system, eyes, and epidermis) exist (e.g. Brüggeman 1985; Rohde et al. 1987; Sopott-Ehlers 1996, 1997; Sopott-Ehlers & Ehlers 1997), but are even more fragmentary than those on spermatology and protonephridia. The Rhabdocoela is still not supported by a clear morphological apomorphy. All rhabdocoels have a ‘pharynx bulbosus’, which they share with the parasitic neodermatans, some prolecithophorans and lecithoepithelians. Therefore it is possible that the ‘pharynx bulbosus’ has originated more than once, and that the homologies should be reconsidered carefully (as suggested by Joffe 1987). A first possible apomorphy could be found in the ultrastructure of the protonephridial system. In all rhabdocoels the terminal cell has a single row of longitudinal ribs (see Rohde 2001). However, this construction is also found in some other taxa (Lecithoepitheliata and Prolecithophora; see Littlewood et al. 1999a; Rohde 2001). A second possible apomorphy based on protonephridial ultrastructure is the lack of a terminal perikaryon, whereas the flame bulb is continuous with the proximal canal cell and without a junction (Type C of Watson & Schockaert 1997; see also Rohde 2001). Moreover, Watson (2001) also proposed the presence of a dense heel on the basal bodies during spermiogenesis as a possible apomorphy for the Rhabdocoela. However, this feature is lost in several taxa within the Rhabdocoela (all kalyptorhynchs, except for some schizorhynchs). For the Kalyptorhynchia two clear apomorphies can be indicated: the presence of a muscular proboscis and the incorporation of the axonemes within the sperm body during spermiogenesis (see Ehlers 1985; Watson 2001). Within the Kalyptorhynchia, a split proboscis and the loss of one axoneme during spermiogenesis (see Watson 2001) characterizes all representatives of the Schizorhynchia. The sister taxon of the Kalyptorhynchia is still unclear, as it forms a polytomy with the Dalytyphloplanida and Mariplanella frisia in all phylogenetic studies based on 18S rDNA. However, the present study shows a relatively high support for a clade uniting M. frisia with the Dalytyphloplanida (see Fig. 1: jackknife 81%). Therefore, this clade most probably is the sister group of the Kalyptorhynchia. The presence of small dense granules, an axonemal spur and a group of longitudinal microtubules (originating from a particular manner of flagellar rotation) in the sperm (see Littlewood et al. 1999a; Watson 2001) are possible apomorphies for the Dalytyphloplanida. However, these features are secondarily lost within some thalassotyphloplanids (in all species formerly included in the Trigonostomidae, which were studied by Watson (2001)) and within the neodalyellids. It is possible that the loss of these features is synapomorphic for a subclade of the Thalassotyphloplanida and, independently, also for a subclade of the Neodalyellida. The Neotyphloplanida consists of a polytomy, including three large freshwater taxa (Temnocephalida, Typhloplanidae, Dalyelliidae), Gaziella sp. and the Thalassotyphloplanida. Littlewood et al. (1999a,b) and Joffe & Kornakova (2001) suggest that a freshwater rhabdocoel clade may exist (Typhloplanidae + Dalyelliidae + Temnocephalida). We did not find such a clade, but there is some support for a clade consisting of the Typhloplanidae and the Dalyelliidae (see Fig. 1: jackknife: 66%). However, with only two species included in the analysis, the dalyelliids (as with the temnocephalids) are very poorly sampled. Based only on our molecular data, all we can say is that the Typhloplanidae (excl. Styloplanella strongylostomoides) and the Dalyelliidae, and probably the Temnocephalida, are each monophyletic freshwater taxa. Nomenclatural implications In this section several new clade names are defined, following phylogenetic nomenclature (De Queiroz & Gauthier 1990, 1992, 1994) and the rules of the draft Phylocode (available at http://www.ohio.edu /phylocode). However, only clades for which strong support ( jackknife > 95%) was found (see also Table 5), and for which we think naming is useful, are defined. To avoid nomenclatural instability, taxa do not receive a converted name when the original nomenclatural types were not included in the analysis. Converted names are indicated with n.c.c. (nomen cladi conversum), whereas completely new clade names receive the indication n.c.n. (nomen cladi novum) following article 9.3 of the Phylocode. Rhabdocoela Ehrenberg, 1831 n.c.c. (stem-based): the most inclusive clade containing Polycystis naegelii Kölliker, 1845 but not Ciliopharyngiella constricta Ax, 1952; Monocelis lineata (Müller, 1774) Oersted, 1844, Nematoplana coelogynoporoides Meixner 1938, Fasciola hepatica Linnaeus, 1758 and Pseudostomum quadrioculatum (Leuckart, 1847) Graff, 1911. Kalyptorhynchia Graff, 1905 n.c.c. (stem-based): the most inclusive clade containing Polycystis naegelii Kölliker, © The Norwegian Academy of Science and Letters 2005 • Zoologica Scripta, 35, 1, January 2006, pp1 – 17 13 18S rDNA phylogeny of Rhabdocoela • W. R. Willems et al. 1845 but not Mariplanella frisia Ax & Heller, 1970 and Provortex balticus (Schultze, 1851) Graff, 1882. Schizorhynchia Meixner, 1928 n.c.c. (stem-based): the most inclusive clade containing Schizochilus marcusi Boaden, 1963 but not Polycyctis naegelii Kölliker, 1845. Dalytyphloplanida n.c.n. (stem-based): the most inclusive clade containing Provortex balticus (Schultze, 1851) Graff, 1882 but not Mariplanella frisia Ax & Heller, 1970 and Polycystis naegelii Kölliker, 1845. Neodalyellida n.c.n. (stem-based): the most inclusive clade containing Provortex balticus (Schultze, 1851) Graff, 1882 but not Proxenetes flabellifer Jensen, 1878. Neotyphloplanida n.c.n. (stem-based): the most inclusive clade containing Proxenetes flabellifer Jensen, 1878 but not Provortex balticus (Schultze, 1851) Graff, 1882. Thalassotyphloplanida n.c.n. (stem-based): the most inclusive clade containing Proxenetes flabellifer Jensen, 1878 but not Castrada lanceola (Braun, 1885) Luther, 1904 and Castrella truncata (Abildgaard, 1789) Hofsten, 1907. Acknowledgements We thank Prof Dr Luc Brendonck (Katholieke Universiteit Leuven) for kindly providing us with the material of Mesostoma thamagai. 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