I n v e i ~ h r a t eBiology 121(4): 357-364. 0 2002 Ainericaii Microscopical Society, Inc
Systematic relationships of Nematomorpha based on molecular and morphological data Christoph Bleidorn,' Andreas Schmidt-Rhaesa,'," and James R. Garey2 I
Dept. of Zoomorphology and Systematics, Faculty of Biology, University of Bielefeld, PO Box 100131, D-33501 Bielefeld, Germany * Dept. of Biology, University of South Florida, Tampa, FL 33620-5150, USA
Abstract. We sequenced the 18s rRNA gene from I 1 nematomorph species from 9 genera and derived hypotheses concerning the sister group of Nematomorpha and relationships within this taxon. The molecular and morphological data are consistent with the monophyly of Ncmatomorpha, a sister-group relationship between Nematomorpha and Nematoda, and a sistergroup relationship between the marine genus Nectonema and all of the freshwater taxa, Gordiida. Hypotheses of relationships within Gordiida support the traditional taxa Gordiidae, Chordodidae, and Chordodinae but reject Parachordodinae and Spinochordodidae. The molecular results differ from those of previous morphological studies by suggesting a reduction of the two tail lobes at the posterior end of males in Chordodinae, monophyly of the genus Paragordiunus, and paraphyly of the genus Chordodes. Additional key words: Nematoda, Nectonema, Gordiida, phylogeny
T h e horsehair worms (Nematomorpha) include about 300 freshwater species (Gordiida) and 5 marine species (Nectonema). They are almost exclusively parasites of arthropods, but leave their hosts for reproduction and early larval development. Originally, nematomorphs had been classified a s nematodes, but they have been treated as a separate taxon since Vejdovsky ( 1886) introduced the name Nematomorpha. The group is considered closely related to Nematoda, and a resemblance to the nematode taxon Mermithidae is sometimes mentioned (Lorenzen 1 985). The nematomorph larva bears an introvert with hooks and stylets which is thought to resemble those of adult priapulids, kinorhynchs, and loriciferans by some authors (Malakhov 1980; Malakhov & Adrianov 1995). A number of potential autapomorphies, such as the mostly subpharyngeal brain, reduction of pharyngeal musculature, and the distinctive larva (SchmidtRhaesa I996), indicate monophyly of Nematomorpha. Gordiida and Nectonema are both monophyletic also; some of the autapomorphies are the subepidermal ventral nerve cord in Gordiida and a blindly ending intestine, natatory bristles, and giant cells in the anterior end in Nectonema (Schmidt-Rhaesa 1996). Most authors agree that nematodes are the sister taxon of Nem-
Author for correspondence. E-mail:
[email protected]
atomorpha (Ehlers et al. 1996; Wallace et al. 1996; Nielsen 2001). Schmidt-Rhaesa (1 998) rcvicwed other hypotheses concerning potential sister taxa of nematomorphs and found none as parsimonious and plausible as the hypothesis of a sister-group relationship o f Nematomorpha and Nematoda. Among Gordiida, the freshwater nematomorphs, 2 1 genera have been described (Schmidt-Rhaesa 2002). One of these genera, Chordodiolus, was recently synonymized with Beatogordius (Schmidt-Rhaesa 2001 ; Schmidt-Rhaesa & de Villalobos 2002). The traditional classification includes 4 families: Gordiidae MAY I9 19 including genera with a so-called postcloacal crescent (Gnrdius and Acutogordius), Spinochordodidae KIRJANOVAI950 including the genus Spinnchordodes, Lanochordodidae KIRJANOVA I950 including the monotypic genus Lanochordodes, and Chordodidae MAY 1919 including all remaining genera. Within Chordodidae, 3 subfamilies are distinguished: Chorclodinae (including all gordiids without a bilobed posterior end in males), Paragordiinae (including Parugordius, Digordius, Progordius, Pseudogordius), and Parachordodinae (including Paruchordodes, Cordionus, Beatogordius, Paragordiunus, and Semigordionus). The aim of this investigation is to test the traditional classification of nematomorphs in a phy logenetic ~ 0 1 1 text-the monophyly of Nematomorpha, the sistergroup relationship to Nematoda, and the sister-group
Bleidorn, Schmidt-Rhaesa, & Garey
358 List of newly sequenced species of nematomorphs. An processing (see Methods).
Table 1.
* indicates a different protocol of DNA extraction and Genbank
Species Chordodes sp. Euchordodes nigromaculatus" Gordionws wolterstotf3* Gordius poran.ensis" Nectonema agile"' Neochordodes occidentalis Pavagordionus dispar Pauagordionus ruutheri Paragordius tricuspidatus Parugoudius vurius Spinochovdodes tellinii
Locality Cameron Highlands, Malaysia Cass, Canterbury Mts., New Zealand
Breitenbach, Schlitz, Germany Cass, Canterbury Mts., New Zealand Passammaquoddy Bay near St. Andrews, N.B., Canada Sandia Site, NE, USA Brcitenbach, Schlitz, Germany River Pegnitz near Furth, Germany Avene-les-Bains, Frances Lancaster Co., NE, USA Avene-les-Bains, France
relationship between Nectonema and Gordiida-using 18s rDNA genes of 1.5 nematomorph species, 11 of which are new sequences. A phylogenetic hypothesis is presented for representatives within Gordiida, which is compared with the traditional classification discussed in the light of morphological characters.
acce\\ion #
AF42 I763 AF42 I764 AF42 1765 AF42 1766 AF42 1767 AF42 1768 AF42 1769 AF42 1770 AF42 1771 AF42 1 772 AF42 1773
tmlis, Scorpiones, Arachnida (X77908); Eurypelnm calljbrnica, Araneae, Arachnida (X l 3457); Panulirus U Y gus, Decapoda, Crustacea (U 191 82); Okanuguna utuhensis, Hemiptera, Insecta (U06478); Tenehrio molitor, Coleoptera, Insecta (X0780 1); Glycera americana, Annelida (U 19.519); Acanthopleum ,japanica, Polyplacophora (X70210); Placopecten rnagellanicus, Bivalvia (X.53899); Phoronis architectu, Phoronida (U3627 1); Methods Antedon serratu, Crinoidea, Echinodermata (D 14357). We sequenced the 1 8 s rRNA gene from I 1 nemaNeighbor-Joining (NJ) and Minimum Evolution tomorph species (Table 1 ) . All species were from the (ME) analyses were carried out i n MEGA version 2.0 collection of A. Schmidt-Rhaesa at the University (Kumar et al. 2000) using Jukes and Cantor and KiBielefeld, Germany. mura two-parameter distance measurements with and DNA was extracted from ethanol-fixed specimens without gamma correction for site variation in substiusing either a phenollchloroform extraction (* in Table tution rate. The gamma shape parameter for NJ and I ) after Hempstead et al. ( I 990) or using a QIAGEN ME analyses was calculated from the data set using DNeasya Tissue Kit. The 18s rDNA gene was PCR Maximum Likelihood (ML) analysis in PAUP. Unamplified in 2 or 3 fragments using coiiserved primers weighted and weighted (2: 1 transversion : transition) (Winnepenninckx et al. 1995; Giribet et al. 1996). PCR Maximum Parsimony (MP) analyses were carried out products were sequenced either by a commercial se- using PAUP version 4.0b8 (Swofford 2000), and Maxquencing service or with a 3 10 Genetic Analyzer (Per- imum Likelihood (ML) analyses were carried out uskin Elmer) after cycle sequencing with dye terminators ing PHYLIP version 3.6 (Felsenstein 1989). Site to site using internal conserved primers. Sequences have been variation in the PHYLIP ML analyses was estimated deposited in Genbank (accession numbers in Table 1). by defining 3 relative rate categories of 1 , 10, and 100 DNA alignments were carried out according to a sec- with respective probabilities of 0.2, 0.6, and 0.2. We ondary structure model using the DCSE software pack- analyzed 1000 bootstrap replicates for NJ trees, 200 age (De Rijk & De Wachter 1993). Sites with gaps were replicates for MP trees, and 100 replicates for ML eliminated from the analyses. The alignment is avail- trees. Trees shown are 50% majority rule consensus able at http:/lchuma.cas.usf.edu/-gareylalignmentsl trees of the bootstrap replicates. alignment.htm1. As outgroup taxa we used (Genbank acScanning electron micrographs (Fig. 4) were of ethcession number): Ascaris suum, Nematoda (AF036587); anol-fixed specimens, dehydrated in a graded ethanol Mermis nigrescens, Nematoda (AF03664 1 ); Xiphinema series, critical point dried, gold sputtered, and viewed ~ivesi,Nematoda (AF036610); Priupulus cuudatus, Pria- in a Hitachi SEM 450 under 15 kV. pulida (X87984); Pycnophyes kielensis, Kinorhyncha Results (U67997); Mucrohiotus hufelandi, Tardigrada (X8 1442); Milnesium tardigradum, Tardigrada (U49909); Thulinia Neighbor-Joining trees of the entire data set using stephaniae, Tardigrada (AF056023); Androctonus aus- Jukes and Cantor and Kimura two-parameter models
Systematic relationships of Nematomorpha
Paragordiunus rautheri Gotdionus woltcr,storfii
I
Nematomorpha
Paragordius tricuspidatus Purqordius warius 100
Nectonema agile
-
77
r
Ecdysozoa
Mrrmbs n ip-wcens
hujelandi Thuliniu .ctephaniue Milnesium turdigraduni
Mucrobiotus
87
Androctonus uu,stralis
95 100
-
Eurypelma californica Panulirus argus Okunagana utahensis
Antedon .wrrufu
Panarthropoda
7Deuterostomia
0.05
Fig. 1. Neighbor-Joining tree o f the entire data set using Kimura two-parameter distances with a gainma shape parameter of 0.34 and bootstrap support values from 1000 replicates. Nematomorpha i s monophyletic and supportcd as a sistcr group of Nematoda within a clade of molting animals (Ecdysozoa).
correcting with different gamma shape parameters not provide bootstrap support for some or the deeper (0.72 as traditionally applied and 0.34 as estimated nodes observed in ME and NJ analyses. Using NJ analyses, a number of different outgroups from the data) resulted in nearly identical topologies (Fig. I ) . Decreasing the shape parameter to 0.2 caused were used. Generally, closely related outgroups such the Neetonemu sequence to appear as the basal mem- as Xiphinema and Priupulus yielded the tree in Fig. 3, ber of the Gordius clade instead of as the basal mem- whereas more distantly related outgroups such as Glyber of Nematomorpha, but the bootstrap value of the cet-a and Antedon yielded trees with Nectonwmu as the Nectonema Got-dius clade was insignificant (34%). basal member of the Gordius clade in all replicates or In all cases, nematomorphs were the sister group to branching between the Gordius clade and the Pamnematodes, appearing within Ecdysozoa as described gordius clade, but with insignificant bootstrap support i n Garey (2001). Removing sequences with the longest (35% and 44%). The phylogenetic relationships according to the 18s branches (Neetonemu, AscariJ, Mermis) in any rDNA gene can be summarized as follows (cf. Fig. 3). combination had no effect on the tree topology. ME, ML, and MP analyses (Fig. 2) were consistent Within Nematomorpha, the marine species Neetonemu with the NJ tree, although MP and ML analysis did agile is the sister group of all analyzed freshwater
+
Bleidorn, Schmidt-Rhaesa, & Garey
360
85
& MP
ME Nematomorpha
& ML
Nematomorpha
Nematomorpha
Nematoda adargidr;:;1
Fig. 2. Schematic representation o f the results of Minimum Evolution (ME), Maximum Parsimony (MP), and Maximum Likelihood (ML) analyses, which all correspond in
Tardigrada
76
Deuterostomia
nematomorphs (Gordiida), which are monophyletic with very high support values. Within Gordiida, there is a cluster of 4 Gordius species, a clade that is not further resolved, but is supported by high bootstrap values. Coi-dius is the sister group to all remaining gordiids. The next split separates Pauagordius, and Gordionus then branches off, followed by Parugordionus. All gordiids with an undivided male posterior end (Chordodinae) form a monophyletic taxon. Within this clade, support values vary and the resolution might therefore be treated with some care. Euchordodes is probably basal. Chordodes is probably paraphyletic.
Discussion Several autapomorphies suggest that Nematomorpha monophyletic (Schmidt-Rhaesa 1996; Nielsen
Arthropoda
Arthropoda
Arthropoda
their topology but differ in bootstrap support values.
is
Nematoda
Scalidophora
64
Spiralia
75
Deuterostomia
Scalidaphora Spiralia Deuterostomia
2001), and also that Nectonerna and Gordiida, the highest ranked sister taxa within Nematomorpha, are each monophyletic (Schmidt-Rhaesa 1996). As the most likely sister group of Nematomorpha, Nematoda is indicated by previously published molecular support, as well as several morphological autapomorphies; together Nematomorpha + Neinatoda constitute the taxon Nematoida SCHMIDT-RHAESA 1996 (SchmidtRhaesa 1998; Zrzavq et al. 1998; Giribet et al. 2000). Our molecular data strongly support these hypotheses. Within Gordiida, the freshwater nematomorphs, systematic analyses beyond traditional classifications have been rare. Schmidt-Rhaesa (2002) reviewed the 2 1 traditional genera and concluded that 7 genera are probably monophyletic and 7 others are probably paraphyletic; the remaining 7 are monotypic. Five genera are potentially synonymous to others and one further
Chordodes morgani Neochordodes occidentaiis Spinochordodes tellinii Chordodes sp. Euchordodes nigromaculatus faragordionus dispar Paragordionus rautheri
c
Fig. 3. The SO% majority rule con-
sensus tree for nematomorpha taxa and the nematode Xiphinema rivesi as an outgroup. Bootstrap support from NJ (above value), MP (middle value), and ML (lower value). Putative morphological autapomorphies of the male posterior end are mapped to five nodes in the tree: 1. tail lobes, 2. postcloacal crescent, 3. precloacal rows of bristles, 4. circumcloacal spines, 5 . reduction of tail lobes.
r
Gordionus violaceus Paragordius tricuspidatus faragordius varius Gordius sp. Gordius paranensis Gordius aquaticus Gordius albopunctatus Nectonema agila Xiphinema rivesi
Systematic relationships of Nematomorpha (monotypic) genus, Chordodiolus HEINZE1934, has been synonymiLed with the genus Beatogordius (Schmidt-Rhaesa 2001 ; Schmidt-Rhaesa & Villalobos 2002). Although not all genera were represented in the analysis, the results of this molecular study are consistent with the traditional classifications of Gordiidae and Chordodidae as introduced by May ( I 919) and Chordodinae as introduced by Heinze (1935). The families Spinochordodidae and Lanochordodidae (Kirjanova 1950) are superfluous, in that they include only one genus each and in the case of Lanochordodes only one species, L. zeravshanicus. Si7inochordode.s tellinii is clearly a member of Chordodinae (Fig. 3). Lanochordodes is probably a synonym of Spinochordodes (Schmidt-Rhaesa 2002). Paragordiinae may also be a redundant taxon, because 3 of the genera included (Digordiu5, Progordius, and Pseudogordius) are probably synonyms of the fourth, Puragordius (Schmidt-Rhaesa 2002). The taxon Parachordodinae, represented in our analysis by Cordionus and Parugordionus, is shown to be paraphyletic. One conspicuous character of Gordiida is the shape of the male posterior end. In several genera, the body is bilobed posterior to the ventral cloacal opening (Fig. 4A,B). In all other genera, a ventral furrow may be present, creating bilateral bulges, but no lobes are present (Fig. 4D). As lobes are lacking in Nectonema and all other outgroup taxa, a bilobed posterior end is a potential autapomorphy of a subtaxon of Gordiida (Schrnidt-Rhaesa 1999). However, this is clearly not supported by molecular data. According to the 18s rRNA gene, tail lobes are an autapomorphy of Gordiida and were 5econdarily reduced in the taxon Chordodinae. The family Gordiidae originally was synonymous with the genus Gordius until Heinze ( 1952) introduced and included the genus Acutogordius. Both genera, Gordius and Acutogordiu share a conspicuous character, the postcloacal crescent (Fig. 4A), which therefore is the autapomorphy of Gordiidae. The relationship of Acutogordius to the probably paraphyletic Gordius (Schmidt-Rhaesa 2002) could not be resolved in our analysis, because no representatives of Acutogordius were included. The 4 Gordius species included in the analysi5 are all very similar. For example, the previously available sequences of G. aquaticus and G. ulbopmctatus differ from each other by a single nucleotide. This is in the range of sequencing error and could be due to a misidentification of one of the specimens. Chordodidae includes all 19 remaining gordiid genera. One potential autapomorphy is the presence of rows or fields of cuticular bristles on the posterior end
36 1
of males (Figs. 3, 4B), occurring most abundantly anterolateral to the cloacal opening, but this character is not unequivocal. Rows of bristles vary much in shape (see Schmidt-Rhaesa 1997) and in fine structure (see Schmidt-Rhaesa 1997; Schmidt-Rhaesa et al. I998), making the assignment of homology questionable. Also, similar structures are present in some Gordius species: G. borisphuenicus SPIRIDONOV 1984, G. difficilis SMITH1994, and G. paranensis CAMERANO 1892 (see Spiridonov 1984; Smith 1994; Schmidt-Rhaesa et al. 2000, respectively). Although representing widely separated geographical regions, the North American species Puragordius varius (LEIDYI85 1) and the European species P. tricuspidatus (DUFOUR1828) are very similar morphologically. Their 18s rRNA gene sequence5 differ from each other in 1 1 positions, comparable to the number of differences among the 4 Gordius species (3-10 positions) and to the number of differences among the 5 Chordodinae sequences. This suggests that P. vurius and P. tricuspidatus may indeed be two distinct species despite their phenotypic similarity. A problematic genus is Paragordionus HEINZE1935, which contains two species (P. dispar and P. vejdovsky) with a unique cuticular structure (Schmidt-Rhaesa 1997) and two further species (P. bohemicus and P. rautheri) that lack this structure, but closely resemble some Gordionus species (Schmidt-Rhaesa 1997). This suggests that the genus might be paraphyletic and some species might belong to Cordionus (Schmidt-Rhaesa 2002). Therefore, finding P. dispar and P. rautheri clearly clustering together in the apparently monophyletic genus Parugordionus was unexpected. One potential autapomorphy of a taxon including Chordodinae, Parugordionus, and Gordionus is the presence of spine5 around the cloacal opening (Figs. 3, 4C). For a taxon Purugordionus Chordodinae, no autapomorphy can be given. All gordiids without tail lobes appear as a monophyletic taxon equivalent to the taxon Chordodinae. Originally this included only the genus Chordudes (Heinm 1935), but other genera without tail lobe5 ruch a5 Euchordodes (Heinze 1 94 I), Neochordodes, and Pseudochordodes (Miralles 1976) were added later. It seems plausible from the molecular data that the lack of tail lobes in Chordodinae is not a primary, but a derived feature. It would be useful to know if tail lobes are formed early or late in the development of .juvenile worms not belonging to Chordodinae. If' they occur late, one might propose heterochrony as the responsible mechanism for the absence of tail lobes in Chordodinae. One of the few genera with a presumably good autapomorphy is the largest genus of Nematomorpha, Chordodes, which includes -90 species (and proba-
+
3 62
Bleidorn, Schmidt-Rhaesa, & Garey
Fig. 4. Gordiid characters. SEM. A. Ventral view of the posterior end of a male of Gordius from Papua New Guinea (Goroka, Eastern Highlands Province) with postcloacal crescent (arrow). B. Posterior end of Gordlonu.c. vio1ac:ru.s (Breitcnbach, Schlitz, Germany) with precloacal rows of bristles (arrow) and circumcloacal spines. C. Circumcloacal spines o f Spinochordodes tellinii from Avene-les Bains (France). D. Ventral view of the posterior end of a male of Spinochoudodes tellinii showing lack of tail lobes.
bly many undescribed species). The autapoinorphy is Dacochordodes bacescui (Spiridonov et al. 1 992) or a characteristic cuticular structure, the so-called Spinochordodes (Schmidt-Rhaesa, unpubl. data). The crowned areoles (Schmidt-Rhaesa 2002). Therefore, crowned areoles of Chordodes are worth it is surprising to find evidence for paraphyly of reinvestigating in more detail. Chordodes in the molecular data, even if the resolution within Chordodinae is not optimal. The North Acknowledgments. Many thanks to several collcagues who American species Chordodes morgani MONTGOMERYaided in the collection of material for this investigation: 1898 is peculiar in that its crowned areoles are very Reinhard Ehrmann (Karlsruhe, Germany), Ben Hanelt (Lininconspicuous (Chandler & Wells 1989; Schmidt- coln, NE), Mr. Hems (Ansbach, Germany), Robert Poulin Rhaesa, unpubl. data) and in some respects resemble (Otago, New Zealand), Will Reeves (Clemson, S C ) , Fred other areolar structures that are present in, e.g., Thomas (Montpellier, France), Peter Zwick (Schlitz, Ger-
Systematic relationships of Nematomorpha many), and people at the St. Andrews Biological Station (New Bi-unswick, Canada).
References Chandler CM & Wells MR 1989. Cuticular features of Chorctode.s morgani (Nematomorpha) using scanning electron micoscopy. Trans. Am. Microsc. Soc. 108: 152-158. De Rijk P & De Wachter R 1993. DCSE: an interactive tool lor sequence alignment and structure research. Comp. Appl. Biosci. 9: 735-740. Ehlers U , Ahlrichs W, Lemburg C, & Schmidt-Rhaesa A 1996. Phylogenetic systematization of the Nemathelininthes (Aschelminthes). Verh. Dtsch. Zool. Ges. 89.1: 8. Felsenstein J 1989. PHYLlP-Phylogeny Inference Package (Version 3.2). University of Washington, Seattle, WA. Carey JR 2001. Ecdysozoa: the relationship between Cycloneuralia and Panarthropoda. Zool. Anz. 240: 321-330. Giribet G, Carranza S, Baguna J, Riutort M, & Ribera C 1996. First molecular evidence for the existence of a Tardigrada + Arthropoda clade. Mol. Biol. Evol. 13: 7684.
Giribet G, Distel DL, Polz M, Sterrer W, & Wheeler W 2000. Triploblastic relationships with emphasis on the acoelomates and the position of Gnathostomulida, Cycliophora, Platyhelminthes, and Chaetognatha: a combined approach of 18s rDNA sequences and morphology. Syst. Biol. 49: 539-563. Heinze K 1935. Uber Gordiiden. Species inquirendae und Neubeschreibungen. Zool. A m . I I I : 23-32. 194 I . Saitenwiirmer oder Gordioidea (Nematomorpha). In: Die Tierwelt Dentschlands. Dahl F, ed., pp. 178. Gustav Fischer Verlag, Jena. 1952. Uber Gordioidea, eine systematische Studie uher Insektenparasiten aus der Gruppe der Nematomorpha. 24. Parasitenkd. IS: 183-202. Hempstead PG, Regular SC, & Ball IR 1990. A method for thc preparation of high-molecular-weight DNA from marine and freshwater triclads. DNA Cell Biol. 9: 5761. Kirjanova ES 1950. Nematomorpha from the river Zeravshan. Trudy Zool. Inst. Akad. Nauk. SSSR 9: 255-280 (in Russian). Kumar S, Tamura K, Jacobsen I, & Nei M 2000. MEGA: Molccular evolutionary genetics analysis, ver. 2. Pennsylvania State University, University Park, and Arizona State University, Tempe. Lorenzen S 1985. Phylogenetic aspects of pseudocoelomate evolution. In: The Origins and Relationships of Lower Invertebrates. Conway Morris S, George JD, Gibson R, & Platt HM, eds., pp. 210-223. Syst. Assoc. Spec. Vol. 28. Clarendon Press, Oxford. Malakhov VV 1980. Cephalorhyncha, a new type of animal kingdom uniting Priapulida, Kinorhyncha, Gordiacea, and a system of Aschelminthes worms. Zool. Zh. 59: 485-499 (in Russian with English summary). Malakhov VV & Adrianov AV 1995. Cephalorhyncha-a New Phylum of the Animal Kingdom. KMK Scientific
363 Press LTD, Moscow, 199 Seiten (in Russian with English summary). May HG 1919. Contributions to the life histories of Gordius rohustus Leidy and Pamgordius varius (Leidy). Illinois Biol. Monogr. 5 : 1-1 19. M i r a k s DAM de 1976. Gordiacea. In: Fauna de agua d u k e de Ia RepGblica Argentina, Vol. 8. Ringuelet RA, ed., 45 pp. Fundaci6n para la Educacih, la Ciencia y la Cultura, Buenos Aires. Nielsen C 2001. Animal Evolution. Oxford University Press, Oxford. 563 pp. Schmidt-Rhaesa A 1996. Monophyly and systeinatic relationships of the Nematomorpha. Verh. Dtsch. Zool. Ges. 89.1: 23. 1997. Nematomorpha. In: SuBw teleuropa, Vol. 4J4. Schwoerbel J & pp. Gustav Fischer Verlag, Stuttgart. 1998. Phylogenetic relationships of the Ncrnatomorpha-a discussion of current hypotheses. Zool. Anz. 236: 203-2 16. 1999. Hypotheses aboul character evolution in Nematomorpha. Zoology 102, Supplement 11: 80 (abstract). 2001. Reinvestigation and reinterpretation o l Chordodiolus cchinatus (Linstow, I90 1 ), a pcculiar nematomorph species. Mitt. Mus. Nat.kd. Berlin, Zool. Reihe 77: 9 1-94, 2002. Are the genera of Nematomorpha monophyletic taxa? Zool. Scripta 3 1 : 185-200. Schmidt-Rhdesa A & de Villalobos LC 2002. Revision ol' the genus Reatogordius (Nematomorpha). 1. African spccies including Chordodiolus echinatus. Annls. Mus. K. Afr. Centr. (Zool.) 290: 1-25. Schmidt-Rhaesa A, Thomas E & in R 1998. Scanning electron microscopy and intrasp variation in Euchordodes nigrornaculatus from New Zealand. J. Helminthology 72: 65-70. 2000. Redescription of Gordius paranensis Camerano, 1892 (Nematomorpha), a species new for New Zealand. J. Nat. Hist. 34: 333-340. Smith DG 1994. A reevaluation of Gordicrs ayuatic~us Montgomery, 1898 (Neinatomoiyha, Gordioidea, G o d idae). Proc. Acad. Nat. Sci. Philadelphia 145: 29-34. Spiridonov SE 1984. Two new species of hairworms of the family Gordiidae. Proc. Zool. Inst. USSR Acad. Sci. 126: 97-101 (in Russian). Spiridonov SE, Pikula ZP, & Drljevic ET 1992. Redescription of Dacochordodes hacescui Capuse, I 966 (Nematomorpha: Chordodidae). Helminthologica (Bratislava) 29: 193- 196. Swofford DL 2000. PAUP". Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. Vejdovsky F 1886. Zur Morphologie der Gordiiden. Z. Wiss. Zool. 43: 369-433. Wallace RL, Ricci C, & Melone G 1996. A cladistic analysis of pseudocoelomate (aschelminth) morphology. Invertebr. Biol. 115: 104-112. Winnepenninckx B, Backeljau T, Mackey LY, Brooks JM,
3 64 De Wachter R, Kumar S, & Garey JR 1995. 18s rRNA data indicate that Aschelminthes are polyphyletic in origin and consist of at least three distinct clades. Mol. Biol. Evol. 12: 1132-1 137.
Bleidorn, Schmidt-Rhaesa, & Garey Zrzavq J, Mihulka S, Kepla P, Be7dEk A, &z Tietr D 1998. Phylogeny of the Metama based on morphological and 1 8 s ribosomal DNA evidence. C l a d i d c s 14: 249-285.
