Research Article |
Corresponding author: Gražina Stanevičiūtė ( grasta@ekoi.lt ) Academic editor: Kira Zadesenets
© 2015 Gražina Stanevičiūtė, Virmantas Stunžėnas, Romualda Petkevičiūtė.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Stanevičiūtė G, Stunžėnas V, Petkevičiūtė R (2015) Phylogenetic relationships of some species of the family Echinostomatidae Odner, 1910 (Trematoda), inferred from nuclear rDNA sequences and karyological analysis. Comparative Cytogenetics 9(2): 257-270. https://doi.org/10.3897/CompCytogen.v9i2.4846
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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.
Echinochasmus , Stephanoprora , Echinostomatidae , karyotype evolution, intermediate host, rDNA, ITS2, 28S
The family Echinostomatidae Looss, 1899 is a heterogeneous group of cosmopolitan, hermaphroditic digeneans. Adult echinostomatids are predominantly found in birds, and also parasitize mammals including man, and occasionally reptiles and fishes (
The karyotypes of more than 20 species of the subfamily Echinostomatinae Looss, 1899 belonging to the genera Echinostoma Rudolphi, 1809, Echinopharyphium Dietz, 1909, Hypoderaeum Dietz, 1909, Neoacanthoparyphium Yamaguti, 1958, Moliniella Hübner, 1939, and Isthmiophora Lühe, 1909 have been described; most species had 2n=20 or 2n=22, except some species (for review, see
The use of molecular approaches to determine phylogenetic relationships of digeneans has grown very rapidly since 1990s and molecular-based studies on echinostomes have been carried out to date (
The present study is mainly focused on comparative analysis of species belonging to the subfamily Echinochasminae. Two regions of rDNA, ITS2 and partial 28S, and karyotype of cercaria of Echinochasmus sp., parasite of the gravel snail Lithoglyphus naticoides (C. Pfeiffer, 1828) are presented there as well as DNA sequences of adult specimen of type-species of Echinochasmus, Echinochasmus coaxatus Dietz, 1909 from the final host Podiceps nigricollis C. L. Brehm, 1831. Morphology of the Echinochasmus sp. cercaria from the same population of L. naticoides was previously described by
The digeneans for this study were obtained from naturally infected hosts. Seven specimens of gravel snail Lithoglyphus naticoides infected with parthenites of Echinochasmus sp. were collected at water reservoir of the dammed up River Nemunas near Kaunas in Lithuania (54°51.38'N, 24°09.08 E’). The specimens of snail Valvata piscinalis (Müller, 1774) infected with parthenites of Echinoparyphium mordwilkoi Skrjabin, 1915 were collected from the River Ūla, Lithuania (54°7.76'N, 24°27.76'E). The ethanol fixed adult specimen of Echinochasmus coaxatus recovered from Podiceps nigricollis in Kherson region (Ukraine) was received from collection of Department of Parasitology, I.I. Schmalhausen Institute of Zoology of NAS of Ukraine. Adult trematodes from Larus melanocephalus (Temminck, 1820) and cercariae from Hydrobia acuta (Draparnaud, 1805) were described as Stephanoprora pseudoechinata (Olsson, 1876) by
Living L. naticoides snails were incubated in 0.01% colchicine in well water for 12–14 h at room temperature and afterward, dissected. The infected tissues from crushed snails were transferred to distilled water for 40–50 min and fixed in a freshly prepared Carnoy’s solution I (Farmer’s solution) composed of 3 parts of 95% ethanol and 1 part glacial acetic acid. Chromosome slides were prepared using air-dried method and analysed after conventional Giemsa staining (
The DNA extraction (without proteinase or lysis buffer treatment) was performed in sterile Tris-borate-EDTA (TBE) buffer. In previous study this method allowed us to extract high quality DNA from tissue of molluscs (
DNA sequences of representative species of the superfamily Echinostomatoidea and outgroup taxa were downloaded from GenBank and included in the phylogenetic analysis and/or pairwise sequence comparisons together with our data. For phylogenetic analyses the sequences were aligned with ClustalW (
Chromosomes of 113 mitotic metaphase spreads from three molluscs revealed that karyotype of Echinochasmus sp. is 2n=20; it consists of one pair of large chromosomes and nine pairs of smaller-size chromosomes. Also, the percentage of aneuploid cells (2n=18–19) was 10.62%. Twelve spreads displaying values lower than modal, represent aneuploidies or (more likely) loss of chromosomes during processing, a technical artefact commonly encountered with the slide preparation method used. The measurements of mitotic chromosomes showed ten chromosome pairs ranging in size from 2.11 to 7.64 μm (Fig.
Morphometric analysis of chromosomes of Echinochasmus sp. Stanevičiūtė, Petkevičiūtė & Kiselienė, 2008.
Chromosome number | Absolute length (mm) | Relative length (%) | Centromeric index | Classification |
---|---|---|---|---|
1 | 7.64*±1.69 | 18.97±1.61 | 37.45±1.64 | sm-m |
2 | 4.99±0.79 | 12.51±0.68 | 10.44± 2.66 | a-st |
3 | 4.72±0.98 | 11.73±0.66 | 23.64±2.25 | st-sm |
4 | 4.46±0.88 | 11.09±0.58 | 14.18±3.62 | st-a |
5 | 3.98±0.78 | 9.89±0.60 | 13.95±4.13 | st-a |
6 | 3.69±0.63 | 9.23±0.64 | 30.39±5.27 | sm |
7 | 3.16±0.53 | 7.89±0.41 | 20.71±2.82 | st |
8 | 2.81±0.40 | 7.05±0.44 | 19.41±2.93 | st |
9 | 2.51±0.28 | 6.33±0.46 | 22.92±5.25 | st |
10 | 2.11±0.38 | 5.29±0.71 | 19.17±4.32 | st |
New sequences from two different regions of nuclear ribosomal DNA were obtained: the 5.8S-ITS2-28S and the 5’ end of the 28S gene, which does not overlap with the previous sequence. Complete nucleotide sequences are available in GenBank (Figs
Phylogenetic ITS2 tree. Maximum likelihood phylogenetic tree based on analysis of ribosomal DNA sequences (5.8S-ITS2-28S). Bootstrap percentages refer to maximum likelihood / neighbor-joing / maximum parsimony analysis. Only bootstrap values above 70% are shown. GenBank accession numbers are indicated before species names. Names of the target species are in bold; their hosts are presented in parentheses. Compressed clades: Fasciola (comprised sequences under GenBank accession numbers AM900370, EF534995, EF612486, JF496715), Echinostoma (AF067850, AF067852, AJ564383, AY168930, EPU58100, ETU58097, ELU58099, GQ463131, GQ463132), Hypoderaeum (AJ564385, GQ463134). Dotted rectangles 1 indicate digeneans whose life cycles include Lymnaeoidea as first intermediate host; dotted rectangle 2 indicates digeneans whose life cycles include prosobranch snails as first intermediate hosts.
Maximum likelihood, neighbor-joining and maximum parsimony analyses of these sequences, including representative species of superfamily Echinostomatoidea, produced identical topology of phylogenetic trees (Figs
Phylogenetic 28S tree. Maximum likelihood phylogenetic tree based on analysis of ribosomal 28S gene DNA partial sequences. Bootstrap percentages refer to maximum likelihood / neighbor-joing / maximum parsimony analysis. Only bootstrap values above 70% are shown. GenBank accession numbers are indicated before species names. Names of the target species are in bold.Compressed clade Fasciola comprised sequences under GenBank accession numbers AY222244, EU025871, EU025872, HM004190). Dotted rectangles 1 indicate digeneans whose life cycles include Lymnaeoidea as first intermediate host; dotted rectangle 2 indicates digeneans whose life cycles include prosobranch snails as first intermediate hosts.
Sequence divergence between S. pseudoechinata and Echinochasmus sp., 1.84% in the 5.8S-ITS2-28S rDNA region and 1.4% in the partial 28S gene, falls within the level of intragenus variability. Both taxa made up a strongly supported clade together with the type-species of the genus Echinochasmus, E. coaxatus. These results imply that macrocercous cercaria of Echinochasmus sp. may be attributed to the genus Stephanoprora Odhner, 1902. According to
The chromosome complement of Echinochasmus sp. with 2n=22 chromosomes gradually decreasing in size and with one-armed elements prevailing are characteristic for species of type-genus Echinostoma (
Idiograms representing the haploid chromosome sets. Idiogram representing the haploid sets of eight species: a Echinochasmus sp. b Episthmium bursicola c Echinochasmus beleocephalus d Echinopharyphium aconiatum e Istmiophora melis f Hypoderaeum conoideum g Sphaeridiotrema globulus h Echinostoma revolutum b, c - data of
Centric fusions could be a possible mechanism for changes in the chromosomal number in this family and in the other digenean groups (
The results of this study indicated that the phylogenetic branching of digeneans is related to the nature of their first intermediate host. Moreover, the mode of karyotype evolution correlates with the intermediate host: a remarkable karyotype variation was detected among species parasitizing prosobranch snails, whereas differences among karyotypes of the species parasitizing lymnaeoid pulmonates snails are not significant.
This research was funded by a grant (No. LEK-10/2010) from the Research Council of Lithuania. The authors also want to express their appreciation to Department of Parasitology, I.I. Schmalhausen Institute of Zoology of NAS of Ukraine for specimen of Echinochasmus coaxatus.