Molecular and morphological evidence for three species of Diplostomum (Digenea: Diplostomidae), parasites of fishes and fish-eating birds in Spain

Abstract

BackgroundRecent 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.MethodsNineteen 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.ResultsIntegrated 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.ConclusionThis first molecular exploration of the diversity of Diplostomum spp. in southern Europe indicates much lower species richness compared with the northern regions of Europe.

Integrative taxonomic approach to the cryptic diversity of Diplostomum spp. in lymnaeid snails from Europe with a focus on the ‘Diplostomum mergi’ species complex

“…However, the species identification of the sequences within ‘Clade Q’ sensu Georgieva et al [ 6 ] is still questionable. The fact that new isolates are being added to this clade ([ 9 , 17 ]; present study) indicates that this lineage has a wider distribution and still requires taxonomic scrutiny. It is worth noting that the metacercaria of ‘ Diplostomum sp.…”

“…parviventosum / spathaceum (JF775727) by Rellstab et al [ 29 ]; and one cercarial isolate ex R. auricularia (JQ665458; isolate RA97) annotated as D. mergi in GenBank but published as D. spathaceum by Behrmann-Godel [ 17 ]. One additional metacercarial isolate belonging to ‘Clade Q’ has been recently sequenced and described by Pérez-del-Olmo et al [ 9 ]. Georgieva et al [ 6 ] also suggested to use temporarily the name D. parviventosum as a label for the four identical sequences (AF419275–AF419278) of Niewiadomska & Laskowski [ 23 ].…”

“…It is worth noting that the metacercaria of ‘ Diplostomum sp. Clade Q’ ex Cyprinus carpio L. from the Ebro Delta in Spain described and sequenced by Pérez-del-Olmo et al [ 9 ] was shown to possess a smaller oral sucker and a shorter holdfast organ compared with the Spanish and Polish (see Niewiadomska [ 27 ]) isolates of D. spathaceum plus a distinctly lower number of excretory granules in the secondary excretory system than the metacercariae of D. spathaceum sensu Niewiadomska [ 27 ]. The cercaria of the single isolate of ‘ Diplostomum sp.…”

“…The first studies addressing integration of morphological assessment and molecular prospecting for species diversity of the genus in Europe resulted in molecular elucidation of the life-cycles of Diplostomum spathaceum (Rudolphi, 1819) (type-species) and D. pseudospathaceum Niewiadomska, 1984 and provided evidence for a substantial unrecognised genetic and morphological diversity, i.e. 15 species-level lineages including three complexes of genetically distinct lineages [ 6 – 9 ].…”

African Diplostomum (sensu Dubois 1961): Minireview on taxonomy and biology

“…Occurrence has been widely adopted by other biological fi elds, the parasitological community has barely used it. In parasitology and Diplostomum in particular, DNA barcoding has been able to reveal diversity and specifi city of metacercariae in hosts [52], disentangle cryptic species [53] and linking life cycle developmental stages [25].…”