BACKGROUND: Recent molecular studies have revealed high species diversity of Diplostomum in central and northern Europe. However, our knowledge of the distribution of Diplostomum spp. in the southern distributional range in Europe of the snail intermediate hosts (Lymnaea stagnalis and Radix spp.) is rather limited. This study aims to fill this gap in our knowledge using molecular and morphological evidence. METHODS: Nineteen fish species and six fish-eating bird species were sampled opportunistically in three regions (Catalonia, Extremadura and Aragon) in Spain. All isolates of Diplostomum spp. were characterised morphologically and molecularly. Partial sequences of the barcode region of the cox1 mitochondrial gene and complete sequences of the ribosomal ITS1-5.8S-ITS2 gene cluster were used for molecular identification of the isolates. RESULTS: Integrated morphological and molecular analyses demonstrated the presence of three species among the larval and adult isolates of Diplostomum spp. sampled in Spain: Diplostomum spathaceum (in fish and birds), D. pseudospathaceum (in birds) and Diplostomum sp. (in fish) referred to as Clade Q sensu Georgieva et al. (Int J Parasitol, 43:57-72, 2013). We detected ten cox1 haplotypes among the isolates of D. spathaceum with only one haplotype shared with adult isolates from central and northern Europe. No specific geographic pattern of the distribution of the novel haplotypes was found. CONCLUSION: This first molecular exploration of the diversity of Diplostomum spp. in southern Europe indicates much lower species richness compared with the northern regions of Europe.

Faculty of Science University of South Bohemia Branišovská 31 370 05 České Budějovice Czech Republic

Institute of Parasitology Biology Centre of the Academy of Sciences of the Czech Republic Branišovská 31 370 05 České Budějovice Czech Republic

Piscifactoría Vegas del Guadiana Gobierno de Extremadura Antigua Ctra N 5 km 391 7 s n C P 06195 Villafranco del Guadiana Badajoz Spain

Unidad de Zoología Marina Institut Cavanilles de Biodiversitat i Biologia Evolutiva Parc Científic Universitat de València PO Box 22085 Valencia 46071 Spain

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Another plea for 'best practice' in molecular approaches to trematode systematics: Diplostomum sp. clade Q identified as Diplostomum baeri Dubois, 1937 in Europe

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

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