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The separation of Clinostomum complanatum Rudolphi, 1814 and Clinostomum marginatum Rudolphi, 1819 has long been unclear. Recent data confirm the validity of the junior species, C. marginatum , by ∼ 1% differences in its 18S rDNA sequences. We collected adults and metacercariae of C. complanatum and C. marginatum and found reliable morphological differences in the genital complex at both developmental stages. In addition, we identified basic morphometrics (distance between suckers, body width) in metacercariae that may be useful for discriminating the species. The morphological differences were supported by the comparison of sequences of internal transcribed spacers of ribosomal DNA and of the mitochondrial gene cytochrome c oxidase I (COI) from 39 specimens. In 36 specimens, the average divergence between the species was 7.3% in ITS and 19.4% in COI sequences. Two specimens from North America and 1 from Europe had sequences that did not allow them to be clearly allied with either species.

Members of the genus Clinostomum Leidy, 1856 are parasites that mature in birds, with occasional reports in humans. Because morphological characters for reliable discrimination of species are lacking, the number of species considered valid has varied by an order of magnitude. In this study, sequences from the DNA barcode region of cytochrome c oxidase I (CO1) and/or internal transcribed spacer (ITS) from specimens from Mexico, Bolivia, Peru, Brazil, Kenya, China and Thailand were analysed together with published sequences from Europe, Africa, Indonesia and North America. Although ITS and CO1 distances among specimens were strongly correlated, distance‐based analysis of each marker yielded different groups. Putative species indicated by CO1 distances were consistent with available morphological identifications, while those indicated by ITS conflicted with morphological identifications in three cases. There was little overlap in sequence variation within and between species, particularly for CO1. Although ITS and CO1 distances tended to increase in specimens that were further apart geographically, this did not impair distance‐based species delineation. Phylogenetic analysis suggests a deep division between clades of Clinostomum inhabiting the New World and Old World, which parallels the distribution of their principal definitive hosts, the Ardeidae.

Higher systematics within the Digenea, Carus 1863 have been relatively stable since a phylogenetic analysis of partial nuclear ribosomal markers (rDNA) led to the erection of the Diplostomida Olson, Cribb, Tkach, Bray, and Littlewood, 2003. However, recent mitochondrial (mt) genome phylogenies suggest this order might be paraphyletic. These analyses show members of two diplostomidan superfamilies are more closely related to the Plagiorchiida La Rue, 1957 than to other members of the Diplostomida. In one of the groups implicated, the Diplostomoidea Poirier, 1886, a recent phylogeny based on mt DNA also indicates the superfamily as a whole is non-monophyletic. To determine if these results were robust to additional taxon sampling, we analyzed mt genomes from seven diplostomoids in three families.To choose between phylogenetic alternatives based on mt genomes and the prior rDNA-based topology, we also analyzed hundreds of ultra-conserved elements (UCEs) assembled from shotgun sequencing. The Diplostomida was paraphyletic in the mt genome phylogeny, but supported in the UCE phylogeny. We speculate this mitonuclear discordance is related to ancient, rapid radiation in the Digenea. Both UCEs and mt genomes support the monophyly of the Diplostomoidea and show congruent relationships within it. The Cyathocotylidae Mühling, 1898 are early diverging descendants of a paraphyletic clade of Diplostomidae Poirier, 1886, in which were nested members of the Strigeidae Railliet, 1919; the results support prior suggestions that the Crassiphialinae Sudarikov, 1960 will rise to the family level. Morphological traits of diplostomoid metacercariae appear to be more useful for differentiating higher taxa than those of adults. We describe a new species of Cotylurus Szidat, 1928, resurrect a species of Hysteromorpha Lutz, 1931, and find support for a species of Alaria Schrank, 1788 of contested validity. Complete rDNA operons are provided as a resource for future studies.

The combined use of morphological and molecular studies allowed for the first time the recognition and description of the adult stage of Clinostomum cutaneum Paperna, 1964 from the grey heron Ardea cinerea L. in Kenya. A redescription of the metacercaria that infect Nile tilapia Oreochromis niloticus niloticus (L.) from the same aquatic environment is also presented. C. cutaneum differs from all other species of Clinostomum Leidy, 1856 in the shape of its uterus. Sequencing the rRNA confirmed the morphological similarity between adults from the grey heron and the metacercarial stage from tilapia, and a level of genetic similarity with the other previously sequenced Clinostomum spp. was observed. The need for a reorganisation of Clinostomum using both morphological and molecular methods is highlighted.

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