Diversity and phylogenetic relationships of European species of Crepidostomum Braun, 1900 (Trematoda: Allocreadiidae) based on rDNA, with special reference to Crepidostomum oschmarini Zhokhov & Pugacheva, 1998

Abstract

Background: Within the genus Crepidostomum Braun, 1900, identification of species and taxonomic decisions made only on the basis of adult morphology have resulted in great problems associated with evaluating actual diversity and validity of species. Life-cycle data, while equal in importance to adult characters, are scarce, controversial or incomplete for most Crepidostomum spp. In this study, rDNA sequences generated from adult and larval Crepidostomum spp. and some other allocreadiid species were analysed to reveal the diversity and phylogenetic relationships of the species and their host range. Detailed morphological description based on light microscopy, SEM tegumental surface topography and genetic data are provided for the poorly known trematode C. oschmarini Zhokhov & Pugacheva, 1998 found in the intestine of two teleost fish species, Barbatula barbatula (L.) and Cottus gobio L.

Results: We characterized 27 isolates of adult and larval parasites. Based on newly obtained 28S and ITS1-5.8S-ITS2 rDNA sequences, new intermediate and final hosts were ascertained, and life-cycles clarified for some allocreadiids. New knowledge on the diversity and phylogenetic relationships of European Crepidostomum spp. was gained. The validity of C. oschmarini was verified based on comparative sequence analysis. Ophthalmoxiphidiocercariae of C. oschmarini were recorded in sphaeriid bivalves Pisidium (Euglesa) casertanum (Poli). Additionally, morphological differences between gravid specimens of C. oschmarini and other related species were observed.

Conclusions: Species of the Allocreadiidae parasitizing fishes in Europe are distributed among two monophyletic genera, Allocreadium and Bunodera, and two paraphyletic Crepidostomum clades. A complex of Crepidostomum metoecus (syn. C. nemachilus), C. oschmarini and Crepidostomum sp. 2 clustered in one clade, and a complex of C. farionis, Crepidostomum sp. 1 and, probably, C. wikgreni in the other. Molecular data indicated that C. oschmarini and Crepidostomum sp. 2 presumably have a wide geographical distribution in Europe. The new data provided evidence that Crepidostomum is a more diverse genus than can be judged from morphological data and host switching in this genus may occur independently of fish-host phylogeny.

Keywords: 28S; Crepidostomum oschmarini; European bullhead Cottus gobio; ITS2 rDNA; Life-cycles; Molecular phylogeny; Morphology; Stone loach Barbatula barbatula; Tegumental topography.

Fig. 1

Crepidostomum oschmarini. a Whole-mount ventral view, ex Barbatula barbatula. b Whole-mount ventral view, ex Cottus gobio. c Terminal genitalia. Scale-bars: a, b, 200 μm; c, 100 μm

Fig. 2

SEM micrographs of the surface topography of the anterior region of the body of Crepidostomum oschmarini. a Ventral view of mature worm. b The constant pattern of 5 papillae on the posterior rim of the oral sucker, 3 symmetrical pairs of papillae in the middle of the anterior rim (white circle) and a group of 8 papillae underneath the sucker rim (black circle). c 3 paired symmetrical papillae (white circle) and the distribution of irregular papillae on the anterior rim. d Interlobular field with marked (white circle) of 6 symmetrical papillae, irregular papillate and non-papillate sensory endings and single papilla on each ventro-lateral lobe (black circles). e Regular pattern of different sizes of the posteriormost, middle and anteriormost pairs of 6 symmetrical papillae on anterior rim. f Regular arrangement of 8 papillae ventro-lateral to the posterior sucker rim (black circle). g Single papilla (black circle) on the surface of a ventro-lateral lobe. h Papillae close to the base of a dorso-lateral lobe. i Ciliated papillate and non-papillate sensory endings on interlobular field. j Ciliated papilla. Abbreviations: ap, anteriormost papilla; c, cilium; cr, cirrus; dll, dorso-lateral lobe; dml, dorso-median lobe; fp, forebody papillae; hb, hindbody; ep, excretory pore; l, lobe; mp, middle papilla; np, non-papillate sensory ending; oa, oral aperture; os, oral sucker; p, ciliated papilla; pp, posteriormost papilla; pr, posterior rim of oral sucker; tr, transverse tegumental ridges; vll, ventro-lateral lobe; vs, ventral sucker

Fig. 3

SEM micrographs of the surface topography of the forebody and hindbody of Crepidostomum oschmarini. a Two symmetrical longitudinal fields of papillae (white circles), the protruded cirrus and the ventral sucker on the forebody. b Ciliated and dome-shaped sensory endings in the two longitudinal fields of papillae. c Ventral sucker with 6 dome-shaped papillae on its rim (white circles). d Radially arranged surface corrugations on the rim of the ventral sucker and the dome-shaped papillae. e Dome-shaped papilla (white circle) on the ventral sucker rim. f Posterior extremity of the body with the terminal excretory pore. g Cobblestone-like protrusions of the body surface. Abbreviations: cp, cobblestone-like protrusion; cr, cirrus; dp, dome-shaped papilla; ep, excretory pore; lp, lateral papilla; p, ciliated papilla; pe, elevation around genital pore; tr, transverse tegumental ridges; vs, ventral sucker

Fig 4

Phylogenetic tree based on Maximum Likelihood analysis of partial sequences of the 28S nuclear rRNA gene. Bootstrap support values lower than 70% are not shown. GenBank accession numbers of sequences in collapsed clades are provided in Table 1. The species sequenced in this study are indicated in bold

Fig. 5

Phylogenetic tree based on maximum likelihood analysis of the ITS2 nuclear rDNA region. Bootstrap support values lower than 70% are not shown. GenBank accession numbers of sequences in collapsed clades are provided in Table 1. The species sequenced in this study are indicated in bold