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. We also thank Mrs Natascha Steffanie (Hasselt
University, Diepenbeek) for her help with PCR and Dr Bonnie
Webster (Natural History Museum, London) for the additional sequences of Promesostoma sp., Einarella argillophyla,
Ciliopharyngiella constricta, Pseudomonocelis ophiocephala and
Trisaccopharynx westbladi. Prof Dr James S. Farris is thanked
for letting us use the Xac program.
WW is supported by a grant of the Flemish Institute for
the Benefit of Scientific and Technological Research in the
Industry (IWT-Flanders). The sampling expeditions were
supported by the project G.0235.02 Phylogeny of Typhloplanoida and Kalyptorhynchia (Platyhelminthes) in a multidisciplinary approach, of the Fund for Scientific Research,
Flanders (FWO). DTJL was funded by a Wellcome Trust
Fellowship (043965/Z/95/Z), and UJ was funded by the
Swedish Research Council.
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