An improved molecular phylogeny of the Nematoda with special emphasis on marine taxa

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Abstract

Phylogenetic reconstructions of relations within the phylum Nematoda are inherently difficult but have been advanced with the introduction of large-scale molecular-based techniques. However, the most recent revisions were heavily biased towards terrestrial and parasitic species and greater representation of clades containing marine species (e.g. Araeolaimida, Chromadorida, Desmodorida, Desmoscolecida, Enoplida, and Monhysterida) is needed for accurate coverage of known taxonomic diversity. We now add small subunit ribosomal DNA (SSU rDNA) sequences for 100 previously un-sequenced species of nematodes, including 46 marine taxa. SSU rDNA sequences for >200 taxa have been analysed based on Bayesian inference and LogDet-transformed distances. The resulting phylogenies provide support for (i) the re-classification of the Secernentea as the order Rhabditida that derived from a common ancestor of chromadorean orders Araeolaimida, Chromadorida, Desmodorida, Desmoscolecida, and Monhysterida and (ii) the position of Bunonema close to the Diplogasteroidea in the Rhabditina. Other, previously controversial relationships can now be resolved more clearly: (a) Alaimus, Campydora, and Trischistoma belong in the Enoplida, (b) Isolaimium is placed basally to a big clade containing the Axonolaimidae, Plectidae, and Rhabditida, (c) Xyzzors belongs in the Desmodoridae, (d) Comesomatidae and Cyartonema belongs in the Monhysterida, (e) Globodera belongs in the Hoplolaimidae and (f) Paratylenchus dianeae belongs in the Criconematoidea. However, the SSU gene did not provide significant support for the class Chromadoria or clear evidence for the relationship between the three classes, Enoplia, Dorylaimia, and Chromadoria. Furthermore, across the whole phylum, the phylogenetically informative characters of the SSU gene are not informative in a parsimony analysis, highlighting the short-comings of the parsimony method for large-scale phylogenetic modelling.

Introduction

The Nematoda are one of the most diverse taxa in the animal kingdom, with estimates ranging from 0.1 to 100 million species (May, 1988, Hammond, 1992, Lambshead, 1993, Coomans, 2000). Free-living species are found in every soil or sedimentary habitat with very few exceptions (e.g. Convey and McInnes, 2005) and are used as indicator species in biodiversity assessments and biomonitoring (reviewed by Lambshead, 2004, Yeates and Boag, 2004, Cook et al., 2005). Nematodes have developed a multitude of parasitic life styles causing numerous human diseases and large financial losses to agriculture and livestock rearing (reviewed by Manzanilla-Lópes et al., 2004). Effective use and control of nematodes requires knowledge of their relationships. Nematodes are also used increasingly as model organisms. Caenorhabditis elegans was the first metazoan organism to have its complete genome sequenced (the C. elegans sequencing Consortium, 1998) and currently over 30 nematode genome sequencing projects are ongoing (Mitreva et al., 2005). However, no sequencing projects are underway for marine nematodes (e.g. Araeolaimida, Chromadorida, Desmodorida, Desmoscolecida, Enoplida, and Monhysterida), largely because it is difficult to collect enough high-quality, species-specific material.

Although life cycles and relationships of nematodes have been studied intensively for over 350 years, the lack of objective criteria for assessing homology of morphological characters has hampered the reconstruction of the phylogeny of the Nematoda. Rarely have marine and terrestrial, animal and plant parasitic species been studied by the same authors. Even where the whole of the phylum has been investigated authors often shoe-horned those groups together for which they did not have much detailed knowledge. Furthermore, the ontogeny and ultrastructure of nematodes is poorly understood and there is a lack of an informative fossil record (e.g. Poinar, 1977, Poinar, 1983, Poinar, 2003). Such difficulties have led to the erection of multiple, at least partially conflicting classifications (De Ley and Blaxter, 2002) that can be grouped into two overall hypotheses.

Chitwood, 1933, Chitwood, 1937, Chitwood and Chitwood, 1950) divided the Nematoda into the Adenophorea (‘gland bearers’) and Secernentea (‘secretors’). The former include virtually all aquatic nematodes (Enoplida and Chromadorida) and selected terrestrial omnivores or plant-feeders (Dorylaimia), the latter group includes almost all parasitic species (Strongylina, Tylenchina, Ascaridina, and Spirurida) and the majority of terrestrial freeliving nematodes (Rhabditina). Lorenzen (1981) followed Chitwood and characterised the classification of the Adenophorea in more detail.

Andrássy (1976) gave each of the two Adenophorean groups, the Torquentia (roughly equivalent to the Chromadorida) and the Penetrantia (roughly equivalent to the Enoplida) the same rank as the Secernentia (≈ Secernentea). However, while ranking them equally, he claimed that the Secernentia evolved from a torquentian ancestor, thus violating the established ranking relationships. Nevertheless, the three-part division found more support overall than the two-part division (Maggenti, 1963, Maggenti, 1970, Gadéa, 1973, Drozdovskii, 1980, Adamson, 1987, Malakhov, 1994).

Advances in molecular-biology techniques allowed an objective, empirical analysis of the evolutionary history of the Nematoda. Blaxter et al. (1998) produced the first molecular phylogenetic framework of the phylum using sequences of the nuclear ribosomal small subunit (SSU). However, their analysis was based primarily on terrestrial and economically important parasitic species such as Dorylaimida, Mermithida, Mononchida, Rhabditida, Trichinellida, and Triplonchida and lacked data from the full range of taxa found in marine habitats (e.g. Araeolaimida, Chromadorida, Desmodorida, Desmoscolecida, Enoplida, and Monhysterida). Further phylum-wide studies (Aleshin et al., 1998, Kampfer et al., 1998, Litvaitis et al., 2000) added more marine species but many major clades remained under-represented (e.g. Enoplida, Chromadorida, Monhysterida, and Desmoscolecida). On the small scale, numerous studies tested molecular markers for the easy identification of pest species. However, these markers, often mitochondrial genes, while able to distinguish between members of the same genus or family, are uninformative for higher level taxonomic studies (e.g. Hyman, 1988, Thomas and Wilson, 1991, Powers et al., 1993, Zarlenga et al., 1998, Hoberg et al., 1999, Watts et al., 1999, Nadler et al., 2000). Recently, De Ley and Blaxter, 2002, De Ley and Blaxter, 2004 updated the classification of the phylum Nematoda using molecular data available from additional species, with morphological data to assist the placement of taxa for which SSU sequences were not yet available. Nevertheless, the system was still based mostly on terrestrial and parasitic taxa.

In this study, we further revise the molecular phylogeny of De Ley and Blaxter (2002) by adding sequences to the nematode SSU data set from previously under-represented marine taxa and from additional terrestrial and parasitic groups. We analysed the phylogeny of 212 nematode taxa and 16 outgroup taxa using two different evolutionary models, Bayesian inference and LogDet-transformed distance analysis. In particular, the addition of sequences from marine taxa was crucial both in resolving the relationships of several major taxa and in affirming the relationships of some previously sequenced species whose phylogenetic positions remained uncertain or controversial.

Section snippets

Coastal sampling

Intertidal sediment and macroalgae samples were taken at several locations in the estuary of Southampton Water, UK, and preserved in 99.7% molecular-grade ethanol. Heavy sediment was removed from the sample by decantation and nematodes were extracted by flotation in Ludox™ 50. Nematodes were mounted individually onto slides for identification (Cook et al., 2005). The first 30 nematodes of seven samples were identified to the lowest taxonomic level possible (Bastian, 1865, Riemann, 1966, Gerlach

Characteristics of different models

The parsimony analyses resulted in topologies that did not correspond to any credible phylogeny, and thus were likely to be driven by phylogenetically uninformative signals or hindered by long-branch-attraction artefacts, to which parsimony is most sensitive (for this and other possible difficulties with using parsimony on rRNA genes for deep phylogeny, see Mallatt et al., 2004). For the Bayesian analysis, majority consensus trees of the 2700 saved trees after burn-in in each of the five

Discussion

Our data bring new resolution to nematode phylogenetics, but there remain areas of uncertainty. Many of the findings are consistent with those of De Ley and Blaxter, 2002, De Ley and Blaxter, 2004. We will focus here on cases where our results differ or provide improved resolution. The Bayesian posterior probabilities and the LogDet bootstrap support values are high for most clades that are consistently reconstructed but low for those recovered by only one of the two analytical methods. While

Conclusions

The addition of 100 new SSU sequences, 46 of which are marine species, has provided additional insights into the phylogeny of the phylum Nematoda. This study presents additional support for (i) the descent of the order Rhabditida from a common ancestor of chromadorean orders Araeolaimida, Chromadorida, Desmodorida, Desmoscolecida, and Monhysterida and (ii) the position of Bunonema close to the Diplogasteroidea in the Rhabditina. The additional data also resolved some previously controversial

Acknowledgments

B.M. thank Dr. Mark Wilkinson, Dr. Peter Foster, Dr. David Gibson, and Dr. Robert Hirt (Natural History Museum) for constructive advice and Dr. Peter Mullin, University of Nebraska, for providing SSU sequences pre-publication. B.M. is indebted to the School of Ocean and Earth Science (University of Southampton), the Department of Zoology (Natural History Museum, London), and the Society of Nematologists for financial support. B.M., N.D., M.A., M.B., A.R., and J.L. acknowledge financial support

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    Present Address: Birgit Meldal, Hutchison/MRC Research Centre, University of Cambridge, Department of Oncology, Addenbrookes Hospital, Hills Road, Cambridge CB2 0XZ, UK.

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