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Molecular Analysis of Nematode Diversity and the Evolution of Parasitism

M. Dorris, P. De Ley and M.L. Blaxter
A thorough and coherent classication of the phylum Nematoda is essential if the evolution of countless phenotypes is to be understood. Here, Mark Dorris, Paul De Ley and Mark Blaxter discuss how the application of molecular phylogenetics is helping to resolve some of the inconsistencies in morphological classication and phylogeny by establishing relationships between free-living and parasitic groups, showing possible patterns underlying the origins of parasitism and placing key nematode species in an evolutionary context for comparative study. Current accepted classication of the phylum Nematoda is based on morphological and ecological traits, primarily in the context of free-living or parasitic phenotypes. The deceptively uniform basic anatomy of nematodes masks a complex pattern of diversity, and estimates of species number within the phylum range from 40 000 to 100 million1. The reliability of nematode morphology in producing a coherent phylogeny has been called into question for several reasons. Not least is the disagreement in resolution at the highest levels evident in systematic studies based on morphology. In ve major phylogenetic representations of the phylum, two classes are recognized: Adenophorea and Secernentea. In two of these analyses2,3, both classes are monophyletic, arising from the same ancestor. The other three phylogenies46 suggest that the Adenophorea are paraphyletic and give rise to the Secernentea. The broad ecology of nematodes within each class supports the latter view. Adenophorea include a wide range of marine, freshwater and soil nematodes, but relatively few parasites of animals and plants, whereas Secernentea occur mostly in terrestrial habitats and include a plethora of parasitic and free-living groups7. This sort of disagreement is echoed by competing analyses at all taxonomic levels within the phylum. A tentative consensus of the current status of morphological phylogeny is shown in Fig. 1. Most nematodes are of microscopic size, and current taxonomy relies largely on their morphological traits as seen with the light microscope. Traits most commonly used are buccal and pharyngeal structure, but other anatomical features such as the cuticle, lip region, intestine, reproductive system, sense organs and tail are also used, as well as life history traits such as parasitic host2. Problems can arise when using morphological traits for phylogeny inference8,9, and these become crucial when the paucity of applicable nematode characters is considered. In addition to observational bias and error, nematodes provide a limited number of characters that can be observed across taxa in relation to the known diversity of species. High levels of homoplasy (parallelism, convergence and reversal; see Box 1 for
Mark Dorris and Mark Blaxter are at the Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh, UK EH9 3JT. Paul De Ley is at the Department of Morphology and Systematics, University of Gent, Belgium. Tel: +44 131 650 6760, Fax: +44 131 650 5450, e-mail: mark.dorris@ed.ac.uk 188

glossary of terms) may be expected to occur in many aspects of nematode morphology visible with light microscopy. Scanning and transmission electron microscopy have revealed many more morphological characters useful in taxonomy and phylogeny10, but their costs have precluded widespread application. A different set of problems in the development of a coherent phylogeny arises from the ecological diversity displayed by nematodes, and the consequent specialization of biologists on one trophic or taxonomic group. Acquiring a holistic picture of the phylum is a daunting task, as different segments of the broad eld of nematology are published in separate journals, and have their own jargon and particular obsessions. For example, parasitologists might be more concerned with the implications of the nematode parasitic phenotype as the causative agent of disease than the classication of ignored animal-parasitic taxa. Finally, the problems of phylogeny reconstruction in nematodes are exacerbated by the lack of any informative fossil evidence11. Therefore, revealing and dating steps in nematode evolution by palaeontological means remains impossible.
Strongylida Spirurida Ascaridida Secernentea Adenophorea Rhigonematida Oxyurida Rhabditida including Caenorhabditis elegans and Strongyloides Diplogasterida Aphelenchida Nematoda Tylenchida Mermithida Trichocephalida Dorylaimida Triplonchida Mononchida Enoplida Monhysterida Chromadorida

Fig. 1. A tentative consensus of the currently accepted systematics of the orders and genera of the phylum Nematoda represented as a cladogram. Adapted from Ref. 25, and based mainly on data from Refs 2 and 3. Parasitology Today, vol. 15, no. 5, 1999

0169-4758/99/$ see front matter 1999 Elsevier Science. All rights reserved. PII: S0169-4758(99)01439-8

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Box 1. Phylogenetic Terms and Conventions The phylogenetics literature abounds with necessary jargon. These terms are not just to confuse the unwary, but serve to dene very closely the qualities and content of items under discussion. These terms and their application are discussed extensively in Ref. 8. Apomorphy A derived character state. Outgroup A group of taxa assumed a priori to lie outside the monophyly of the taxa under analysis; used to give diBranch The segment linking one node with another, or a rection to determination of character change. node with a terminal taxon. Paralogy Homology having arisen through gene duplication. Characters Variable features that can assume one of a Paraphyletic A taxonomic group which does not include number of different states. all the descendants of an ancestral taxon. Clade A (monophyletic) group of organisms related by Phylogram A representation of a phylogeny where evodescent from a common ancestor. lutionary relatedness is shown by both branching order and Cladistics A phylogenetic approach that only admits to bia distance measure. furcations in lineages (no polytomies or reticulate evoluPhylogeny A hypothesis of the relationships of organisms. tion) and has explicit rules for their derivation. Plesiomorphy The ancestral character state. Cladogram A cladistic representation of a phylogeny, Polyphyletic A taxonomic group which derives from >1 whereby only the branching order is displayed. ancestral taxon. Homology Common ancestry of two genes (characters, Polytomy (unresolved node) A node that gives rise to >2 genes, positions). descendent taxa. Homoplasy, parallelism or convergence Independent Resolved phylogeny A phylogeny in which all relationderivation of a character state in two lineages. ships are represented by bifurcations. Ingroup The taxa under analysis. Reversal Change of a character from an apomorphic to a Monophyletic A clade where all the taxa derive from a plesiomorphic state. single common ancestor, and which includes all the deRooted phylogeny A phylogeny in which, by use of an scendents of that ancestor. outgroup, the last common ancestor of the clade of taxa Node A branchpoint in a tree (a presumed ancestral under consideration can be placed. taxon). Synapomorphy A shared derived character state (in refOrthology True homologues: homologues that have erence to a phylogenetic hypothesis). arisen through speciation of their host genomes, rather than Unrooted phylogeny A phylogeny where no outgroup is specied. by gene duplication within a genome.

A new framework Box 2. Inferring Phylogenetic Trees Molecular phylogenetic methods Many different criteria can be used to infer phylogenetic trees from morphologiallow comparison of disparate taxa cal or molecular data. All methods are based on two processes: an algorithm for using the same metric: the evolunding trees and a criterion for selecting the best ones. It is expedient to apply tion of a single conserved molecule. all the criteria to each data set and to test the derived trees for consistency and statistical support (see below). The three major criteria used are: This approach sidesteps some of the problems of the denition of homNeighbour joining (NJ) ology and is synergistically compatNJ analysis yields a point estimate of a minimum evolution tree based on data ible with morphological systematics. transformed into a pairwise distance matrix (in this method, the algorithm for nding the tree and the criteria for assessing its quality are combined). The use of molecular markers certainly brings its own problems but, Maximum likelihood (ML) in general, the mode of evolution ML analysis uses an explicit model of evolution (direction and probability of of DNA sequences is better undercharacter change) to derive the tree most likely to have occurred given the data. stood than that of morphological Many different trees can be built and tested. traits, and can be modelled with some Maximum parsimony (MP) condence. This allows alternative MP is a criterion for selecting an optimal tree based on the principle that the analytical tools to be used and pertree requiring the least number of changes in character states is more favoured. mits calculation of statistical supMany different trees can be built and tested. Among methods for testing the internal statistical support for the inferred tree port for the phylogenies produced is the bootstrap. Bootstrap resampling rebuilds a number (usually >100) of (Box 2). An important consideration model data sets based on the test set (by sampling with replacement) and reis that the rates of phylesis (the genanalyses them with the chosen criteria. The percent retention of nodes in the set eration of taxa; speciation) and xof bootstrap trees is a strong indication of their robustness. Bootstrap values ation of molecular change must be >65% are considered robust. of the same order. Thus, a rapidly evolving DNA segment should be used to examine the relationships between species in a Ribosomal RNA genes as phylogenetic markers genus, and a very conserved segment for interordinal, All molecular studies rely on the assumption that the or interphylum relationships. It should be borne in sequences from the taxa being compared are ortholomind that the phylogeny derived from a single molgous, although this assumption is often speculative, esecule might not faithfully reect the history of all pecially for distantly related taxa12. Orthologues are homologues that have arisen through phylesis (organspecies studied, and that information from multiple ismal speciation), rather than gene duplication within a unlinked genetic loci will give more robust estimates. genome. Only orthologues can reect the historical reAcquisition of multiple data sets from independent lationships of a species. Ribosomal RNA (rRNA) genes genes, and analyses using multiple methods with apare found in all organisms and retain a basic shared funcplication of statistical tests to the resultant trees, are imtion. They have been used extensively for the derivation portant components of building testable hypotheses.
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of phylogenies from species to kingSequence variation dom level. They are present in multiHighly ple copies per genome: Caenorhabditis variable elegans has about 55 sets of rRNA genes13: the number present in other nematodes is not known but is likely to be similar. Molecular drive results Highly in the concerted evolution of the conserved rRNA loci through intrachromosoNTS ITS1 ITS2 NTS SSU (18S) 5.8S LSU (28S) mal homogenization14,15. Thus, each Size (bp) ~1000 1700 150 3400 copy of the rRNA cistron is identical ~1000 and can be regarded as orthologous. Fig. 2. The ribosomal RNA (rRNA) cistron. Sizes are approximate and not to scale. The The structure of an rRNA cistron is rRNA cistron is present in ~55 directly repeated copies per nematode genome: each illustrated in Fig. 2. cistron comprises the small subunit gene (SSU; also called 18S), the internal transcribed For the rRNA gene products there spacer 1 (ITS1), the 5.8S gene, ITS2 and the large subunit gene (LSU or 28S). An exis a robust biological model of secondternal nontranscribed spacer (NTS) separates each transcribed cistron. The relative ary structure and a large database of rate of sequence variation observed between taxa is shown schematically above the sequences, permitting alignment of cistron and illustrates that the SSU and LSU sequences are the most conserved, folnew sequences constrained by the lowed by the ITS regions. The NTS region is the most variable in length and sequence. base pairing of the model. The rDNA In addition to the gross variation between the segments of the rRNA cistron, the genes sequences and intergenic spacer rethemselves comprise a mosaic of highly conserved and variable regions. gions provide a rich source of molecular characters for developing a phylogenetic framework for nematodes16. Nematodes one of the major orders within Adenophorea. Some scenhave evolved and diverged over a long period of time arios similar to this relationship have been proposed and require relatively invariant sequences for analysis. previously on morphological grounds46, but these did Small subunit (SSU) rRNA genes at around 1700 base not address the probable paraphyly of the Chromadorida. pairs are easy to amplify and sequence and contain An independent SSU analysis conrms the paraphyly highly conserved domains. For organisms evolving of the Chromadorida30. Indeed, analysis of all the availover a shorter time period, rRNA internal transcribed able nematode SSU sequences (ie. from the three puband terminal nontranscribed spacer sequences have lished accounts25,29,30) yields trees with the same topolbeen used17. Therefore, it is possible to use the riboogy as found by Blaxter et al.25, suggesting that the somal cistron to develop phylogenies of both distantly hypothesis is robust (M.L. Blaxter, unpublished). and closely related organisms. It is also possible to amFive major clades are identied. Two strictly adenoplify rDNA segments reliably from single, xed speciphorean clades (I and II) are strongly supported: both mens1820: this allows the analysis of museum collections contain plant-parasitic taxa, and clade I includes the Triand thus even rare or extinct species. chocephalida (Trichinella and Trichuris) and the Mermithida (insect parasites). Three major clades (IIIV) are The deep phylogenetic structure of nematodes found within the Secernentea. These clades do not correrevealed by SSU analysis spond to the classic divisions in which taxa are grouped The SSU rRNA gene has proved to be highly inforprimarily by trophic ecology. Rather, the plant- and mative for resolving the deep structure of the Nemaanimal-parasitic orders are crown taxa integrated within toda16,21. The relationships of the phylum to other animal a radiation of free-living stem groups. The animalphyla have been debated and contested for a century. parasitic orders Ascaridida (ascarids including Toxocara Recently, SSU rDNA data have been used to show that and Ascaris), Oxyurida (pinworms and relatives), Spithe Aschelminthes, a taxon that includes a number of rurida (larial nematodes and relatives) and Rhigoworm-like phyla, is polyphyletic22. There is even evidence nematida (millipede parasites) form the rst clade (III) for a close relationship between nematodes, arthropods within Secernentea. The closest free-living species to the and other moulting animals23,24. The most comprehenparasites of clade III are the Plectidae and Teratosive molecular phylogeny to date has been established cephalidae, both of which occur predominantly in freshusing SSU rDNA sequences from taxa sampled across water sediments and moist soils. The other two clades the entire phylum25. For the rst time, animal-parasitic, include free-living taxa corresponding loosely to the plant-parasitic and free-living taxa have been compared Cephalobina (IV) and Rhabditina/Diplogasterina (V) of directly. The results are summarized in Fig. 3. classic taxonomy. However, in addition to the microAlthough roughly similar to previous hypothbivorous cephalobes, clade IV also includes the animaleses2,3,7,2628, this independent overview of the phylum parasitic Strongyloides (traditionally considered to be highlights a number of paraphyletic taxa, suggests new rhabditid), the entomopathogen Steinernema, the mainly relationships between previously unconnected taxa, and fungivorous Aphelenchida and the plant-parasitic Tylenseparates some taxa previously believed to be closely chida (including the root-knot and cyst nematodes). related. The Nematoda is seen to arise from an adenoThe proximity of cephalobes to tylenchs has little morphorean ancestry: the classic split into Adenophorea and phological support and hence contradicts mainstream Secernentea is not supported, contrary to a less-extensive opinion27, but is supported by a synapomorphic pattern analysis of nematode SSU sequences29, where tree topolof embryonic axis specication31. Clade IV can be split ogy was probably distorted by lower numbers and diverinto two subclades, although extreme differences in insity of included taxa24. Instead, the Secernentea are found ferred branch length (and thus evolutionary rates) among to arise from within the Chromadorida, traditionally the taxa sampled make this separation less certain, and
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Rapidly evolving rDNA segments illuminate local phylogenies Rapidly evolving segments of the rDNA cistron can be used to derive phylogenies for more limited parts of the nematode tree. For example, loop regions of the SSU and large subunit (LSU) from ascaridoids yielded phylogenies that differ from accepted morphological classications, but agree with minority views based on alternative interpretations of the morphological data32, and are supported by analysis of full-length SSU sequences33. 5.8S rRNA sequences have been used to compare the orders Strongylida, Rhabditida, Spirurida and Tylenchida34. Nematodes of the order Strongylida have remarkably similar SSU sequences, despite their morphological divergence. The genetic distance between the most widely separated taxa sampled (from different families) is of the same order as that between species of the genus Caenorhabditis, and much smaller than that between species of Strongyloides, suggesting an overall lack of synchronicity between morphological and molecular rates of divergence in nematodes. Thus, to discern the relationships of Australian marsupial strongylids, the internal transcribed spacer 2 (ITS2) region was used35 (see Fig. 2). The marsupial parasites have a very restricted host distribution (herbivorous wombats and kangaroos) compared with placental mammals. Are the marsuFig. 3. The phylogenetic structure of the Nematoda revealed by analysis of full-length pial parasites monophyletic, as has small subunit (SSU) rDNA sequences25. Full-length sequences were determined and been proposed from morphological aligned using a secondary structure model for nematode SSU ribosomal RNA (rRNA). data, or are they the stem group from The alignment was analysed by several phylogenetic methods, including maximum parwhich eutherian parasites have arisen, simony and neighbour joining (Box 2)25. The numbers above the branches indicate bootor have there been multiple introducstrap support for each branching event. Five major clades are identied and labelled I tions/transfers from placental mamto V (clade IV is split into a and b); the trophic ecology of each clade is indicated by mals or other host groups36? The ITS2 an icon. The Chromadorida is paraphyletic, and the relationships of the Chromadorida and the Secernentea (clades III, IV and V) remain unclear. data strongly suggest a polyphyletic origin and have brought into question the inferred direction of character morphologically intermediate species remain to be change in one of the central characters used for the morsampled. Clade V joins many families of free-living phological analysis (the structure of the ovipositor) (Fig. rhabditids with the animal-parasitic Strongylida, a re4). These data also conict with a simple hostparasite colationship supported by putative synapomorphies in speciation model, and illustrate the propensity of nemabuccal structure and male caudal morphology. todes for morphological and ecological homoplasy36. The By this molecular framework, animal parasitism is ITS regions are variable enough to separate even crypinferred to have arisen independently at least six times, tic species37, and can be used as a molecular ngerprint and plant parasitism three times. Current research is to identify eggs and larvae in pathology specimens20. focused on placing additional parasitic taxa in this framework, and investigating the placement of various Mode and tempo of the evolution of parasitism problematical taxa whose affinities are unclear. For exBranch length data from molecular phylogenies can ample, within the Rhabditina (clade V) there is great be related to evolutionary time if a model of molecular interest in dening the basal groups that gave rise to evolution is applied that assumes clock-like accumulation the radiation of taxa seen today. Some candidate taxa of genetic change38,39. This assumption of a molecular (eg. Teratorhabditis) have been sequenced but have such clock allows inferred branch lengths to be read as time long inferred branch lengths that a unique branching intervals, ie. a time axis can be placed on the tree. Fossils order is not supported statistically21. are required to calibrate and validate the clock, and their
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clade V) are similar to those seen between tetrapod classes: the Nematoda appears to be old and diverse16,21. The genetic divergence between taxa in the Strongylida (and also between the four parasitic orders in clade III) is remarkably low. This pattern suggests either a relative slowdown in molecular evolutionary rates, correlated with the adoption of the parasitic mode of life, or an increase in the relative rate of morphological evolution (and thus recent radiation), or both. One possibility is that the molecular evolutionary rate is correlated with generation time41: many animal parasites have longer generation times and shorter inferred branch lengths in the SSU phylogenies. However, comparative analysis of branch length with generation time has thus far shown no signicant association (A. Webster, A. Purvis and M.L. Blaxter, unpublished). If we assume that rates of molecular change (per unit evolutionary time) are not radically different between animal parasites and their free-living relatives, the conclusion can be drawn that both the Strongylida and clade III have radiated very rapidly to colonize the majority of vertebrate species (particularly terrestrial species). This could have been achieved through two mechanisms: co-speciation (parallel phylesis), or colonization followed by speciation alternatives that can be tested by looking at patterns of congruence between host and parasite phylogenies42. At higher levels (ordinal to familial), our current model is of explosive horizontal transfer to multiple hosts after initial independent inventions of parasitism. Intimate co-speciation of parasites and their hosts might also be less common than expected, as suggested by both the Australian strongyle study described above35 and our own research within the genus Strongyloides (M. Dorris, M. Viney and M.L. Blaxter, unpublished).

Fig. 4. Analysis of the origins and evolution of strongylid parasites of marsupials using the ribosomal internal transcribed spacer 2 (ITS2) sequences. ITS2 sequences (essentially from Ref. 35, with additional hookworm and Oesophagostomum sequences) were retrieved from GenBank and aligned. The alignment was analysed for phylogenetic content using maximum parsimony and neighbour joining methods, as described in Ref. 35. Regions where the alignment was doubtful (where there were large apparent insertions or deletions in one or more sequences) were excluded from the analysis. The trees obtained were assessed for support using the bootstrap resampling procedure (100 replications). The tree gured is from a neighbour joining analysis with the Kimura 2parameter distance correction. Trees derived from maximum parsimony analysis are congruent with this one. Percentage bootstrap support (where >50%) is given beside each node. Marsupial parasites are indicated in blue, whereas eutherian mammal parasites are in black. Clades containing taxa with Y-shaped ovejectors are indicated by heavy red lines. From this analysis it would appear that the marsupial parasites group into three clades, two having only marsupial-parasitic members. Oesophagostomoides roots robustly with eutherian parasites from the Chabertiidae. Thus, marsupial parasites are not monophyletic, and may have arisen from a eutherian-parasitic ancestor. The pattern of change of the morphology of the ovejector suggests that a Y-shaped form has evolved in parallel in two clades, the Chabertiidae and the Cloacininae.

absence in nematodes invalidates clock assumptions for the SSU nematode phylogeny. In addition, extreme rate differences in inferred accumulation of changes are seen between taxa25. These rate differences are even more problematic in that they can cause signicant artefacts in the building of trees40. Of note here is that the distances between genera within the Rhabditina (in
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Evolution of animal-parasitic life histories It has been proposed that the earliest animal-parasitic nematodes were lumenal dwellers that fed on intestinal microora, and that the primitive transmission mode was cutaneous penetration2,7. However, a single mechanism for the evolution of parasitism is an unnecessary and unlikely simplication, especially as numerous kinds and degrees of associations with other animals occur and these associations must also have been abundant in the past. The SSU data suggest that animal parasitism has evolved independently at least six times, from distinct
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ancestors. (A number of lesser-known parasitic taxa remain to be analysed, and thus this estimate may rise.) An alternative hypothesis might be that the parasitic trait has been secondarily lost in most lineages, but this can be rejected by considerations of evolutionary parsimony. The emerging data suggest an association between invertebrate and vertebrate parasitism, with invertebratepathogenic and parasitic clades lying basal to major vertebrate-parasitic ones. Clade III includes numerous arthropod gut parasites, suggesting that oral ingestion might be ancestral in this clade. However, in clade IV, a cutaneous invasion strategy is predicted to be ancestral. The sister taxa of both the Strongylida and Strongyloides are entomopathogens (Heterorhabditis and Steinernema, respectively), which enter their hosts by cutaneous invasion25. Heterorhabditis has an external tooth with which it can puncture the host cuticle, while Steinernema invades through body orices. Steinernema and Heterorhabditis also exemplify the complexity of nematode life histories. Both are obligate pathogens of insects and share a similar unusual trophic ecology: active entry and release of a toxic bacterial symbiont that kills the host well before the completion of the nematodes life cycle. Remarkably, this adaptation has arisen independently in the two genera, graphically illustrating the pitfalls in inferring phylogeny from obvious ecology. Thus, the new phylogenies might lead to a more cautious perspective on the evolution of parasitism, in which each taxon must be analysed in its own right, and where adaptations have apparently been channelled more by ecological opportunity than by historical constraints. They will also be essential for the application of the comparative method to reveal the evolutionary dynamics of complex traits, such as tissue migration43, facultative parasitism and covariation between parasite and host44,45.
Acknowledgements We thank our colleagues in the nematode phylogenetics study (Jac Vaneteren, Kelley Thomas, Leo Liu and James Garey) for their fruitful collaboration, and many colleagues in the nematology and parasitology community for nematode samples. Andrew Read helped focus the manuscript. MD is funded by the NERC, PDL by the Belgian NFvWO and MB by the Darwin Trust.
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