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Background  Metacercariae of  Diplostomum  are important fish pathogens, but reliable data on their diversity in natural fish populations are virtually lacking. This study was conducted to explore the species diversity and host-parasite association patterns of  Diplostomum  spp. in a large riverine system in Europe, using molecular and morphological data.    Methods  Twenty-eight species of fish of nine families were sampled in the River Danube at Nyergesújfalu in Hungary in 2012 and Štúrovo in Slovakia in 2015. Isolates of  Diplostomum  spp. were characterised morphologically and molecularly. Partial sequences of the ‘barcode’ region of the cytochrome  c  oxidase subunit 1 ( cox 1) and complete sequences of the nicotinamide adenine dinucleotide dehydrogenase subunit 3 ( nad 3) mitochondrial genes were amplified for 76 and 30 isolates, respectively. The partial  cox 1 sequences were used for molecular identification of the isolates and an assessment of haplotype diversity and possible host-associated structuring of the most prevalent parasite species. New primers were designed for amplification of the mitochondrial  nad 3 gene.    Results  Only lens-infecting  Diplostomum  spp. were recovered in 16 fish species of five families. Barcoding of representative isolates provided molecular identification for three species/species-level genetic lineages,  D .  spathaceum ,  D .  pseudospathaceum  and ‘ D .  mergi  Lineage 2’, and three single isolates potentially representing distinct species. Molecular data helped to elucidate partially the life-cycle of ‘ D .  mergi  Lineage 2’. Many of the haplotypes of  D .  spathaceum  (16 in total),  D .  pseudospathaceum  (15 in total) and ‘ D .  mergi  Lineage 2’ (7 in total) were shared by a number of fish hosts and there was no indication of genetic structuring associated with the second intermediate host. The most frequent  Diplostomum  spp. exhibited a low host-specificity, predominantly infecting a wide range of cyprinid fishes, but also species of distant fish families such as the Acipenseridae, Lotidae, Percidae and Siluridae. The  nad 3 gene exhibited distinctly higher levels of interspecific divergence in comparison with the  cox 1 gene.    Conclusions  This first exploration of the species diversity and host ranges of  Diplostomum  spp., in natural fish populations in the River Danube, provided novel molecular, morphological and host-use data which will advance further ecological studies on the distribution and host ranges of these important fish parasites in Europe. Our results also indicate that the  nad 3 gene is a good candidate marker for multi-gene approaches to systematic estimates within the genus.    Electronic supplementary material  The online version of this article (10.1186/s13071-017-2518-5) contains supplementary material, which is available to authorized users.

Exploring the diversity of Diplostomum (Digenea: Diplostomidae) in fishes from the River Danube using mitochondrial DNA barcodes