Three species of bucephalid digeneans are known in European freshwater habitats. In this study parthenitae of Rhipidocotyle campanula (Dujardin, 1845) and R. fennica Gibson, Taskinen & Valtonen, 1992, infecting unionid bivalves, and adult Bucephalus polymorphus von Baer, 1827 from perch (Perca fluviatilis L.) were investigated using karyological analysis and DNA sequencing. Our previously published data on genetic characteristics of parthenitae of B. polymorphus from Dreissena polymorpha Pallas were used for comparative analysis. Ribosomal DNA sequences (ITS2 and 28S rDNA) were used to estimate the phylogenetic relationships of the three bucephalid species. Very close phylogenetic affinity between investigated species was revealed; the sequence difference between the two species of Rhipidocotyle Diesing, 1858 (3.78% based on 28S) was comparable with intergeneric differences observed in comparisons of B. polymorphus with R. campanula and R. fennica (3.43% and 4.49% based on 28S, respectively). A high degree of similarity was noted in karyotype structure of the two species of Rhipidocotyle. The diploid chromosome sets consist of 14 bi-armed chromosomes with the first pair of metacentric elements markedly larger than the remaining chromosomes. This chromosome set structure is also specific to B. polymorphus. One specimen of Anodonta anatina L. was infected with tetraploid R. fennica (4n = 28). On the basis of karyotype characters and molecular data, species of the genus Rhipidocotyle cannot be recognised as more closely related to each other than to B. polymorphus. Our findings of Lithuanian and Ukrainian populations of unionid mussels infected with R. fennica provide evidence that this species occurs not only in Finland but also in Central and Eastern Europe. Previous reports of B. polymorphus in unionids in these regions are equivocal because of possible confusion with R. fennica.

Petkevi ci ut_ e, R., Stun z_ enas, V., Stanevi ci ut_ e, G., Zhokhov, A.E. (2015). European Phyllodistomum (Digenea, Gorgoderidae) and phylogenetic affinities of Cercaria duplicata based on rDNA and karyotypes. 44,[191][192][193][194][195][196][197][198][199][200][201][202] Genetic markers of some European Gorgoderidae species, including potential adults of Cercaria duplicata, were obtained and used to clarify phylogenetic affinities within the genus Phyllodistomum and to verify conflicting data existing on their life cycles. Molecular data and karyotype, 2n = 18, provide further support for ascription of C. duplicata to the Gorgoderinae. Sequences of C. duplicata form a robustly supported major clade, phylogenetically distinct from other known gorgoderid species in both ITS2-and 28S-based phylogenetic trees. The molecular data revealed no match between C. duplicata and any species of Phyllodistomum, including adults found in the experimental studies. One of them, P. elongatum, showed no differences from type species P. folium. Other, P. angulatum, forms a robustly supported clade, which is closely related to P. macrocotyle clade in all phylogenetic trees. This study supports the concept that only P. macrocotyle is a parasite of Dreissena polymorpha among the phyllodistomes and life cycle described for the type species P. folium by Sinitsin (1905) can apparently be discounted. Previously reported low host specificity of P. folium was justified. Adults of P. folium were detected in eight teleost species from five families and four orders. Cystocercous cercariae of P. folium were recorded in sphaeriid bivalves of the genus Sphaerium and Pisidium. According to our molecular data, P. simile, parasite of bullhead, must be regarded as synonym of P. folium. Phyllodistomum umblae is most closely related to P. folium in the all phylogenetic analyses. Molecular phylogenies support a presumption that Phyllodistomum species with cystocercous cercariae developing in sphaeriid bivalves should be regarded as Phyllodistomum sensu stricto. The results reveal a clear need for reconsideration of the knowledge on gorgoderid life cycles based on experimental studies and re-evaluation of the validity of some nominal Phyllodistomum species. Corresponding author: Romualda Petkevi ci ut_ e,

The present work represents the first karyological and molecular characterisation of Sphaerium solidum, a rare European clam. Specimens of S. solidum were collected in Lithuania and Hungary. The modal diploid chromosome number found in both populations was 2n=30. Small, biarmed B chromosomes were found in 42.3% of cells studied in clams from Lithuania and in 11.8% of cells in clams from Hungary. Comparative analysis revealed no significant (P<0.05) interspecific differences in chromosome morphology of S. solidum and that of previously studied S. corneum. DNA sequence analyses of S. solidum showed no interpopulation differences in ITS1; moreover, only one site was different from ITS1 of S. corneum. However, differences in mitochondrial 16S sequence of S. solidum were revealed: two haplotypes in Lithuania and three in Hungary were identified. The genetic characteristics revealed in this study do not support ascription of S. solidum and S. corneum to different subgenus, Cyrenastrum and Sphaerium s. str., respectively. Comparative cytogenetic analysis disclosed that the chromosome morphology could be conserved in some sphaeriid species during speciation despite the fact that most other species in this family undergo radical karyotypic differentiation.

Genetic data were used to examine the diversity in some allocreadiid trematodes. Nuclear ribosomal DNA (ITS2 and partial sequences of 5.8S and 28S) was sampled from sexual adult and 'larval' stages. From these and previous reference datasets phylogenetic trees were constructed. The results uncovered genetically distinct lineages within Bunodera luciopercae (Müller, 1776), suggesting that the two Palaearctic subspecies, B. l. luciopercae and B. l. acerinae Roitman & Sokolov, 1999, and Nearctic B. luciopercae from Perca flavescens may represent distinct species with a restricted host-specificity. Identical rDNA was revealed for the sexual adult of B. l. acerinae and 'larval' B. luciopercae described by Wiśniewski (1958). An unexpected match between the rDNA sequences of adult B. l. luciopercae and 'larval' Allocreadium isoporum (sensu Wiśniewski, 1958) was also detected. The adult A. isoporum (Looss, 1894) differs significantly from the 'larval' A. isoporum, the level of rDNA sequence divergence between them (8.6 % for 5.8S-ITS2-28S sequences and 6.26% for 28S) being consistent with the level expected for intergeneric variation. These results revealed the possible existence of a cryptic species complex within the nominal species B. luciopercae and a clear need for reconsideration of some of the accepted, but largely untested, tenets regarding allocreadiid life-cycles.

Two populations of Sphaerium corneum were sampled from River Vilnel and small pond in Vilnius, Lithuania. The chromosomes were studied using conventional Giemsa staining and karyometric analysis. Inter-and intra-individual variation in the diploid chromosome numbers was revealed and two different sources of chromosome variability were identified: B chromosomes and the structural changes of chromosomes of the basic (A) set. The chromosome set of the more common karyotypic form, 2 n = 30, found in both populations, consists of all biarmed metacentric and meta-submetacentric chromosomes of gradually decreasing size. Small, biarmed, mitotically unstable B chromosomes were found in the cells of this karyotypic form. Specimens with 2 n = 36 were found only in pond. No B chromosomes were detected in their cells. The karyotype is characterized by presence of two pairs of medium telocentrics and four pairs of small subtelocentrics. The remaining chromosomes are biarmed. Robertsonian fusions appear to be involved in formation of two karyotypic forms of S. corneum . DNA sequence analyses showed that ITS1 is identical in both karyotypic forms. On the other hand, differences in 16S sequence were revealed and two haplotypes, corresponding to two karyotypic forms, were identified. The present study opens new perspectives in establishing speciesspecific characters for confident identification of Sphaerium species and provides insights to the genetic intraspecific variability and possible mechanisms of speciation.

BackgroundHost-specificity patterns are not well-defined for trematodes of the genus Phyllodistomum Braun, 1899. The Eurasian ruffe, Gymnocephalus cernuus L., has been recorded as a definitive host for Phyllodistomum folium (Olfers, 1816), P. angulatum Linstow, 1907 and P. megalorchis Nybelin, 1926 and as the type-host for P. pseudofolium Nybelin (1926). A wide range of other host fishes have been recorded for these species as well. All present host records have been based on light microscopy and the life-cycles of P. pseudofolium, P. angulatum and P. megalorchis are unknown. The validity of P. pseudofolium and P. megalorchis require verification. In this study, rDNA sequences generated from adult Phyllodistomum spp., as well as from larval stages developing in Pisidium amnicum Müller, were analysed to establish the real number of Phyllodistomum species utilizing G. cernuus, and to associate larvae with the corresponding adult forms.ResultsPhylogenetic analyses of adult and larval stages of Phyllodistomum spp. based on ITS2 and partial 28S rDNA data allowed the confirmation of the validity of P. pseudofolium. A macrocercous cercaria, known as Phyllodistomum sp. from P. amnicum is genetically identical to adult P. pseudofolium. Phyllodistomum megalorchis obtained from its type-host, Lota lota L., showed no genetic differences from P. angulatum parasitizing Sander lucioperca L. In our analysis, P. pseudofolium, P. angulatum and P. macrocotyle formed a highly supported clade despite the fact that these species appear to be associated with distinct patterns of first intermediate host identity and cercarial morphology. Some morphological differences between gravid specimens of P. pseudofolium and P. angulatum were observed and their SEM tegumental surface topography is described.ConclusionsThe results lead us to the perception that macroevolutionary host switching in the genus Phyllodistomum is independent of host phylogeny. This study suggests strict host-specificity (oioxeny) for P. pseudofolium using one first intermediate host species (P. amnicum) and one definitive host species (G. cernuus). Phyllodistomum megalorchis is to be regarded as a synonym of P. angulatum. The close phylogenetic relatives, P. pseudofolium and P. angulatum, can be differentiated by morphological traits, the micromorphology and tegumental surface topography of these two species is intended to provide useful data for their identification and support the use of such features as a valuable taxonomic criterion. Molecular data showed that G. cernuus is a definitive host for two species: the oioxenous P. pseudofolium and the euryxenous P. folium.

Chromosome sets of two North American sphaeriid species, Sphaerium rhomboideum Say, 1822 and S. occidentale Lewis, 1856, were studied using conventional Giemsa staining and karyometric analysis. Pronounced karyological divergence of congeners was revealed. The diploid number of 2n ¼ 44 was reported for S. rhomboideum and this is the first record of a diploid species in the highly polychromosomic Nearctic sphaeriid fauna. The karyotype was characterized by medium-sized and small chromosomes, which decreased in size gradually from 5.77 to 1.9 mm. Biarmed chromosomes with medially and submedially located centromeres predominated, but six pairs of subtelo-telocentric elements were also observed in the karyotype. The estimated mitotic chromosome number for S. occidentale ranges from 189 to 213, but most of the cells examined contained about 204-209 chromosomes. A first attempt to karyotype a polyploid sphaeriid was made. It was revealed that the comparatively large and middle-sized chromosomes could be grouped in four, so the karyotype presumably evolved through tetraploidization. The small chromosomes formed the large fraction, about 137. Due to their similar and indistinct morphologies, it was impossible to arrange them into subgroups with confidence. Revealed karyological characteristics are discussed with reference to the existing phylogenetic interpretations of the evolutionary history of the Sphaeriinae.

BackgroundWithin 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.ResultsWe 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.ConclusionsSpecies 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.