The phylum Platyhelminthes is a diverse group of flatworms that includes parasites with serious impacts on human health, animal husbandry, aquaculture and wildlife management. Here we present degenerate primers for the barcode region of the mitochondrial cytochrome c oxidase I (COI) gene in flatworms. Although amplicons were obtained from a wide taxonomic range in the Cestoda and Trematoda, COI fragments from many taxa in these classes did not amplify. Primers specific to trematodes in the family Diplostomidae were also developed. Amplification success was much higher with diplostomid-specific primers and sequences were obtained from 504 of 585 specimens of Diplostomum and Tylodelphys. Sequences from the barcode region resolved all specimens to the species level, with mean divergence between congeners of 19% (3.9-25%). Because many of our specimens were small, we initially amplified part of the nuclear small subunit ribosomal (r) RNA gene to evaluate the quality and quantity of DNA in our specimens. Short sequences (~380 nt) of this gene were recovered from most specimens and can be used to distinguish specimens at the family level and often the generic level. We suggest that rRNA genes could be used to screen samples of completely unknown taxonomy, after which specific COI primers could be used to obtain species-level identifications.

Diplostomoid metacercariae parasitize freshwater fishes worldwide and cannot be identified to species based on morphology. In this study, sequences of the barcode region of cytochrome c oxidase subunit 1 (CO1) were used to discriminate species in 1088 diplostomoids, most of which were metacercariae from fish collected in the St. Lawrence River, Canada. Forty-seven diplostomoid species were detected, representing a large increase in known diversity. Most species suggested by CO1 sequences were supported by sequences of internal transcribed spacer (ITS) of rDNA and host and tissue specificity. Three lines of evidence indicate that physiological incompatibility between host and parasite is a more important determinant of host specificity than ecological separation of hosts and parasites in this important group of freshwater fish pathogens. First, nearly all diplostomoid species residing outside the lens of the eyes of fish are highly host specific, while all species that occur inside the lens are generalists. This can be plausibly explained by a physiological mechanism, namely the lack of an effective immune response in the lens. Second, the distribution of diplostomoid species among fish taxa reflected the phylogenetic relationships of host species rather than their ecological similarities. Third, the same patterns of host specificity were observed in separate, ecologically distinctive fish communities.

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The systematics of Diplostomum species, common intestinal parasites of piscivorous birds, has long been problematic, owing to phenotypic plasticity and the paucity of morphological features that are often subject to age-and host-induced variation. We sequenced the ITS1-5.8S-ITS2 regions of the rDNA from adult Diplostomum huronense, Diplostomum indistinctum, and Diplostomum baeri obtained from experimentally infected ring-bill gulls (Larus delawarensis) and compared them with partial ITS1 sequences from several species of Diplostomum in GenBank. The three North American species were distinguishable on the basis of ITS sequences. Sequences from D. huronense differed from those of D. indistinctum at 12 sites in ITS1 and 4 sites in ITS2, supporting morphological and morphometric data that indicate the two are distinct species. Sequences of D. huronense and D. indistinctum differed from those of D. baeri at 27 and 24 sites, respectively, in ITS1 and 15 and 12 sites, respectively, in ITS2. Phylogenetic analysis of partial ITS1 sequences revealed that the North American and European species of Diplostomum formed separate groups, with the former being basal to the latter. The results indicated that D. huronense and D. indistinctum from North America are distinct from Diplostomum spathaceum and other similar species from Europe. Furthermore, sequences from specimens identified as D. baeri from North America differed from those of D. baeri from Europe by 3.8% in ITS1 (23 sites). While morphologically similar, the two are not conspecific. Sequences of the North American species have been deposited in GenBank (AY 123042-123044).Résumé : La systématique des espèces de Diplostomum, parasites intestinaux communs des oiseaux piscivores, a toujours été problématique à cause de leur plasticité phénotypique et de la rareté des caractères morphologiques qui sont souvent soumis à des variations dues à l'âge et à l'hôte. Nous avons procédé au séquençage des régions ITS1-5.8S-ITS2 du gène d'ADNr de Diplostomum huronense, de Diplostomum indistinctum et Diplostomum baeri adultes provenant de goélands à bec cerclé (Larus delawarensis) infectés expérimentalement et nous avons comparé les séquences obtenues à des séquences partielles d'ITS1 de plusieurs espèces de Diplostomum de la banque génétique GenBank. Les trois espèces nord-américaines se distinguent par les séquences de leurs ITS. Les séquences de D. huronense diffèrent de celles de D. indistinctum à 12 sites sur ITS1 et à 4 sites sur ITS2, ce qui corrobore les données morphologiques et morphométriques qui indiquent qu'il s'agit de deux espèces distinctes. Les séquences de D. huronense et de D. indistinctum diffèrent de celles de D. baeri à 27 et 24 sites sur ITS1 et à 15 et 12 sites sur ITS2, respectivement. L'analyse phylogénétique des séquences partielles D'ITS1 a révélé que les espèces nord-américaines et européennes de Diplostomum forment deux groupes distincts, le premier groupe étant plus ancien. Les résultats indiquent que D. huronense et D. indistinctum d'Amérique du Nord so...