The taxonomic history of members of the 37-collar-spine group within the genus Echinostoma has been very confused. We obtained DNA sequence data from the nuclear rDNA ITS1, 5.8S and ITS2 of 7 nominal species belonging to this group, Echinostoma trivolvis (Cert, 1914), E. revolutum (Frolich, 1802), E. caproni Richard, 1964, E. acaproni arasasingam et nl. 1952, E. paraelzsei Lie & Basch, 1967, two African isolates, E. sp.I and E. sp.II, and of one 28-collar-spined echinostome, E. holtense (,4sada, 1926). Five of the eight species n;ere clearly distinguishable using ITS data. Sequences from the remaining three tasa, E. cnpl olzi, E. sp.ll and E. liel mere identical to one another and the group containing these laxa was distant from other 3'7-collar-spine species on a phylogenetic tree. E. tvizrolzris and E. pavaensei form a second, but less distinct group within the 35-collar-spine group. The resolution obtained using DNA sequencing will assist in the current reclassification of the group. It also provides a model for future work on sibling species.

Nuclear rDNA ITS sequence variation in the trematode genus Echinostoma : an aid to establishing relationships within the 37-collar-spine group

Differences in a ribosomal DNA sequence of morphologically indistinguishable species within the Hypodontus macropi complex (Nematoda: Strongyloidea)

In just over a decade, the use of molecular approaches for the recognition of parasites has become commonplace. For trematodes, the internal transcribed spacer region of ribosomal DNA (ITS rDNA) has become the default region of choice. Here, we review the findings of 63 studies that report ITS rDNA sequence data for about 155 digenean species from 19 families, and then review the levels of variation that have been reported and how the variation has been interpreted. Overall, complete ITS sequences (or ITS1 or ITS2 regions alone) usually distinguish trematode species clearly, including combinations for which morphology gives ambiguous results. Closely related species may have few base differences and in at least one convincing case the ITS2 sequences of two "good" species are identical. In some cases, the ITS1 region gives greater resolution than the ITS2 because of the presence of variable repeat units that are generally lacking in the ITS2. Intraspecific variation is usually low and frequently apparently absent. Information on geographical variation of digeneans is limited but at least some of the reported variation probably reflects the presence of multiple species. Despite the accepted dogma that concerted evolution makes the individual representative of the entire species, a significant number of studies have reported at least some intraspecific variation. The significance of such variation is difficult to assess a posteriori, but it seems likely that identification and sequencing errors account for some of it and failure to recognise separate species may also be significant. Some reported variation clearly requires further analysis. The use of a "yardstick" to determine when separate species should be recognised is flawed. Instead, we argue that consistent genetic differences that are associated with consistent morphological or biological traits should be considered the marker for separate species. We propose a generalised approach to the use of rDNA to distinguish trematode species.

The Use and Implications of Ribosomal DNA Sequencing for the Discrimination of Digenean Species

European Lymnaeidae (Mollusca: Gastropoda), intermediate hosts of trematodiases, based on nuclear ribosomal DNA ITS-2 sequences.

We examined the relative merits of mitochondrial DNA loci and ribosomal DNA internal transcribed spacers for their use in prospecting for cryptic species of platyhelminth parasites. Sequence divergence at ITS1 and ITS2 was compared with divergence at 2 mtDNA loci (NADH dehydrogenase-1 and cytochrome c oxidase I) between closely related species of trematodes and cestodes. Both spacers accumulated substitutions substantially more slowly than mtDNA, which clearly shows a higher level of divergence among species relative to intra-specific variation. Besides a slow rate of substitution, other caveats that may be encountered when using ITS sequences as a prospecting marker are discussed. In particular, we note recent studies that suggest the existence of substantial levels of intra-individual variation in ITS sequences of flatworms. Because it is likely that closely related species share this phenomenon, it may confound the detection of cryptic species, especially if small sample sizes are studied. Although potential limitations of mtDNA are also recognized, the higher rate of evolution and smaller effective population size of this marker increases the probability of detecting diagnostic characters between cryptic species.

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Kim Luton

Papers

Comparisons of ribosomal internal transcribed spacers from two congeneric species of flukes (Platyhelminthes: Trematoda: Digenea)