title: Echinostoma 'revolutum' (Digenea: Echinostomatidae) species complex revisited: species delimitation based on novel molecular and morphological data gathered in Europe.
abstract: BACKGROUND
  The systematics of echinostomes within the so-called 'revolutum' group of the genus Echinostoma, which encompasses the type-species E. revolutum and a number of morphologically similar species, has long been controversial. Recent molecular studies indicate the existence of more species than previously considered valid, thus stressing the need for wider taxon sampling from natural host populations. This is especially true for Europe where morphological evidence indicates higher species diversity than previously thought, but where molecular data are virtually lacking. This gap in our knowledge was addressed in the present study through an integration of morphological and molecular approaches in the investigation of a dataset with larger taxonomic and geographical coverage.
  
  
  METHODS
  More than 20,000 freshwater snails belonging to 16 species were collected during 1998-2012 from various localities in eight countries in Europe. Snail screening provided representative larval isolates for five species of the 'revolutum' group, identified by their morphology. Adult isolates for four species recovered from natural and experimental infections were also identified. Partial fragments of the mitochondrial nad1 and 28S rRNA genes were amplified for 74 and 16 isolates, respectively; these were analysed together with the sequences of Echinostoma spp. available on GenBank.
  
  
  RESULTS
  Delineation of the European Echinostoma spp. was carried out based on molecular, morphological and ecological data. The large-scale screening revealed infections with five Echinostoma spp., including one new species: E. revolutum (sensu stricto), E. miyagawai, E. paraulum, E. bolschewense and Echinostoma n. sp. The newly-generated nad1 sequences from Europe fall into six distinct, well-supported, reciprocally monophyletic lineages corresponding to the species identifications based on morphology; this was corroborated by the 28S rDNA sequences. The analyses of the total nad1 dataset provided evidence for 12 monophyletic groups and five singletons, which represent seven described/named species and ten cryptic species-level lineages of Echinostoma.
  
  
  CONCLUSIONS
  We conclude that nad1 should be the first choice for large-scale barcode-based identification of the species of the 'revolutum' group. Our study provides a comprehensive reference library for precisely identified isolates of the European species and highlights the importance of an integrative approach for species identification linking molecular, morphological and biological data.
authors.affiliation: Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, 370 05, České Budějovice, Czech Republic. simona.georgieva@gmail.com.|Faculty of Science, University of South Bohemia, Branišovská 31, 370 05, České Budějovice, Czech Republic. simona.georgieva@gmail.com.
authors.forename: Simona
authors.initials: S
authors.lastname: Georgieva
authors.affiliation: Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, 370 05, České Budějovice, Czech Republic. faltyn@paru.cas.cz.
authors.forename: Anna
authors.initials: A
authors.lastname: Faltýnková
authors.affiliation: Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, 370 05, České Budějovice, Czech Republic. rebeccabrown1200@hotmail.com.|College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK. rebeccabrown1200@hotmail.com.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Brown
authors.affiliation: Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, 370 05, České Budějovice, Czech Republic. isa.blasco.costa@gmail.com.|Natural History Museum of Geneva, P.O. Box 6134, CH-1211, Geneva, Switzerland. isa.blasco.costa@gmail.com.
authors.forename: Isabel
authors.initials: I
authors.lastname: Blasco-Costa
authors.affiliation: Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, 370 05, České Budějovice, Czech Republic. soldanova@paru.cas.cz.
authors.forename: Miroslava
authors.initials: M
authors.lastname: Soldánová
authors.affiliation: Komenský Museum, Horní nám, 7, 750 11 Přerov 2, Přerov, Czech Republic. sitko@cmail.cz.
authors.forename: Jiljí
authors.initials: J
authors.lastname: Sitko
authors.affiliation: Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, 370 05, České Budějovice, Czech Republic. tscholz@paru.cas.cz.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Scholz
authors.affiliation: Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, 370 05, České Budějovice, Czech Republic. aneta.kostadinova@uv.es.
authors.forename: Aneta
authors.initials: A
authors.lastname: Kostadinova
mesh_terms: D000818:Animals; D004449:Echinostoma / Q000033:anatomy & histology / Q000145:classification* / Q000235:genetics*; D005060:Europe; D005618:Fresh Water; D006239:Haplotypes; D006790:Host-Parasite Interactions; D010802:Phylogeny; D012908:Snails / Q000469:parasitology; D013045:Species Specificity