Diversity and Phylogenetic Relationships of European Species of \u3ci\u3eCrepidostomum\u3c/i\u3e Braun, 1900 (Trematoda: Allocreadiidae) Based on rDNA, with Special Reference to \u3ci\u3eCrepidostomum oschmarini\u3c/i\u3e Zhokhov & Pugacheva, 1998

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 analyzed 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 phylogen

Comments on species divergence in the genus Sphaerium (Bivalvia) and phylogenetic affinities of Sphaerium nucleus and S. corneum var. mamillanum based on karyotypes and sequences of 16S and ITS1 rDNA

Chromosome, 16S and ITS1 rDNA sequence analyses were used to obtain reliable diagnostic characters and to clarify phylogenetic relationships of sphaeriid bivalves of the genus Sphaerium. The species studied were found to be diploid, with modal number 2n = 28 in S. nucleus and 2n = 30 in S. corneum var. mamillanum. Small, biarmed, C- negative B chromosomes were found in all studied populations of both species. Karyological and molecular markers revealed no differences between S. corneum s. str. and S. corneum var. mamillanum. No intraspecific differences were found in the basic karyotype of S. nucleus. Molecular analyses, however, uncovered three genetically distinct ITS1 lineages: one comprised of samples from Lithuania, Slovakia, and Russia, another from Czech, and a third from Ukraine. Additionally to known 16S haplotype from Ukraine, three new 16S haplotypes of S. nucleus were detected: one in the samples from Lithuania and Russia, one in Slovakian and one in Czech population. In the ITS1 phylogenetic tree, all branches of S. nucleus clustered in one clade. In the 16S phylogenetic tree, however, the haplotype of Czech S. nucleus formed a separate branch, distant from three other haplotypes of S. nucleus. Molecular results indicate that in the context of the Evolutionary Species Concept the S. nucleus mor-phospecies may represent a complex of separate taxa, however referring on the Biological Species Concept the genetic lineages could represent the intraspecific variability

Phylogenetic relationships of some species of the family Echinostomatidae Odner, 1910 (Trematoda), inferred from nuclear rDNA sequences and karyological analysis

The family Echinostomatidae Looss, 1899 exhibits a substantial taxonomic diversity, morphological criteria adopted by different authors have resulted in its subdivision into an impressive number of subfamilies. The status of the subfamily Echinochasminae Odhner, 1910 was changed in various classifications. Genetic characteristics and phylogenetic analysis of four Echinostomatidae species – Echinochasmus sp., Echinochasmus coaxatus Dietz, 1909, Stephanoprora pseudoechinata (Olsson, 1876) and Echinoparyphium mordwilkoi Skrjabin, 1915 were obtained to understand well enough the homogeneity of the Echinochasminae and phylogenetic relationships within the Echinostomatidae. Chromosome set and nuclear rDNA (ITS2 and 28S) sequences of parthenites of Echinochasmus sp. were studied. The karyotype of this species (2n=20, one pair of large bi-armed chromosomes and others are smaller-sized, mainly one-armed, chromosomes) differed from that previously described for two other representatives of the Echinochasminae, E. beleocephalus (von Linstow, 1893), 2n=14, and Episthmium bursicola (Creplin, 1937), 2n=18. In phylogenetic trees based on ITS2 and 28S datasets, a well-supported subclade with Echinochasmus sp. and Stephanoprora pseudoechinata clustered with one well-supported clade together with Echinochasmus japonicus Tanabe, 1926 (data only for 28S) and E. coaxatus. These results supported close phylogenetic relationships between Echinochasmus Dietz, 1909 and Stephanoprora Odhner, 1902. Phylogenetic analysis revealed a clear separation of related species of Echinostomatoidea restricted to prosobranch snails as first intermediate hosts, from other species of Echinostomatidae and Psilostomidae, developing in Lymnaeoidea snails as first intermediate hosts. According to the data based on rDNA phylogeny, it was supposed that evolution of parasitic flukes linked with first intermediate hosts. Digeneans parasitizing prosobranch snails showed higher dynamic of karyotype evolution provided by different chromosomal rearrangements including Robertsonian translocations and pericentric inversions than more stable karyotype of digenean worms parasitizing lymnaeoid pulmonate snails

Hidden diversity in European Allocreadium spp. (Trematoda, Allocreadiidae) and the discovery of the adult stage of Cercariaeum crassum Wesenberg-Lund, 1934

DNA sequences for adult and larval Allocreadium spp. from their natural fish and molluscan hosts were generated. Phylogenetic analyses based on two molecular markers (ITS2 and 28S rDNA) yielded unexpected results regarding the diversity and life cycles of European species. It was found that specimens morphologically consistent with the concept of Allocreadium isoporum (Looss 1894) form two different species-level genetic lineages. For now, the morphological differences between the specimens belonging to different genetic lineages are not discernible; they can infect the same fish species at the same or different localities. However, the species differ in their life-cycle patterns, specifically in terms of larval stages and first intermediate host specificity. Based on molecular markers, the tailed ophthalmoxiphidiocercaria developing in Pisidium spp. was associated with a sexual adult A. isoporum from Alburnus alburnus, Barbatula barbatula and Rutilus rutilus. Representatives of another genetic lineage, recovered from R. rutilus and Scardinius erythrophthalmus, turned out to be conspecific with the enigmatic European larval trematode Cercariaeum crassum Wesenberg-Lund, 1934, from the sphaeriid bivalve Pisidium amnicum. This finding requires the recognition of the cryptic species Allocreadium crassum

<i>Sphaerium</i> spp. subjected to molecular phylogenetic analysis with information of their host, locality and GenBank accession numbers.

<p><i>Sphaerium</i> spp. subjected to molecular phylogenetic analysis with information of their host, locality and GenBank accession numbers.</p

Phylogenetic tree obtained from ITS1 sequences of nuclear rDNA and based on the analysis of 520 sites.

<p>Bootstrap support given for maximum likelihood analysis (bootstrap replications = 1000, complete deletion of gaps/missing data). Bootstrap support values lower than 70% are not shown. Names of the target species are in bold. <i>Pisidium dubium</i> and <i>P</i>. <i>variable</i> were included as outgroups.</p

Average number of nucleotide differences between ITS1 dataset sequences of closest related groups of <i>Sphaerium</i> spp. with pairwise deletion of gaps/missing data and inclusion of all substitutions (transitions and transversions).

<p>Average number of nucleotide differences between ITS1 dataset sequences of closest related groups of <i>Sphaerium</i> spp. with pairwise deletion of gaps/missing data and inclusion of all substitutions (transitions and transversions).</p

Measurements (mean±SD) and classification of modal diploid (A) chromosomes of <i>Sphaerium corneum</i> var. <i>mamillanum</i>.

<p>Measurements (mean±SD) and classification of modal diploid (A) chromosomes of <i>Sphaerium corneum</i> var. <i>mamillanum</i>.</p

Comments on species divergence in the genus <i>Sphaerium</i> (Bivalvia) and phylogenetic affinities of <i>Sphaerium nucleus</i> and <i>S</i>. <i>corneum</i> var. <i>mamillanum</i> based on karyotypes and sequences of 16S and ITS1 rDNA - Fig 2

<p><b>Mitotic metaphases and respective karyotypes of <i>Sphaerium nucleus</i> with different numbers of B chromosomes: a, 2n = 28 + 4B, and b, C-banded chromosomes, 2n = 28 + 8B.</b> Scale bars = 10 μm.</p