See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/271707812 On the systematic position of Collyricloides massanae Vaucher, 1969 (Platyhelminthes: Digenea) with notes on distribution of this trematode species Article in Parasitology Research · February 2015 DOI: 10.1007/s00436-015-4333-2 · Source: PubMed CITATIONS READS 9 107 5 authors, including: Gerard Kanarek Grzegorz Zaleśny Polish Academy of Sciences Wrocław University of Environmental and Life Sciences 24 PUBLICATIONS 180 CITATIONS 69 PUBLICATIONS 460 CITATIONS SEE PROFILE SEE PROFILE Agnieszka Czujkowska Zoo Warsaw 10 PUBLICATIONS 43 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: Molecular Epidemiology of Bartonella View project Heligmosomoides taxonomy View project All content following this page was uploaded by Grzegorz Zaleśny on 05 February 2015. The user has requested enhancement of the downloaded file. Parasitol Res DOI 10.1007/s00436-015-4333-2 ORIGINAL PAPER On the systematic position of Collyricloides massanae Vaucher, 1969 (Platyhelminthes: Digenea) with notes on distribution of this trematode species Gerard Kanarek & Grzegorz Zaleśny & Agnieszka Czujkowska & Jiljí Sitko & Philip D. Harris Received: 12 December 2014 /Accepted: 20 January 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Abstract The systematic position of the Collyricloides Keywords Trematoda . Microphalloidea . Collyriclidae . massanae, a rare cyst-dwelling parasite, located on intestinal Collyricloides . Molecular phylogeny wall of European birds and rodents, have always been contro- versial. Based on newly obtained sequences of the 28 sDNA of C. massanae from avian and rodent host from Central Europe, and on the previously published sequences of several Introduction genera and families among Microphalloidea, we evaluate its taxonomic position and the phylogenetic relationships within The digenean Collyriclum faba is a moderately well-known the genera Collyriclum Kossack, 1911 and Collyricloides parasite found in subcutaneous tissue cysts in a variety of bird Vaucher, 1969 which form the family Collyriclidae Ward, species. The systematic position of this parasite has been doubt- 1917. In the cladogram, C. massanae appears among the ful for many years: Odhner (1914) placed the genus in his family Pleurogenidae, forming a clade with Gyrabascus Troglotrematidae, while Ward (1917) regarding the isolated po- amphoraeformis (Modlinger, 1930) and Cortrema sition of Collyriclum among monostomes, created the new fam- magnicaudata (Bykhovskaya-Pavlovskaya, 1950). We reject ily Collyriclidae Ward, 1917. Validity of the family was the commonly accepted placement of Collyricloides as the confirmedbyHarrah(1922), but many authors of this period sister genus to Collyriclum within the Collyriclidae. Besides, (e.g., Baer 1932;Witenberg1932; Wallace 1935) continued to we present and discuss the unusual records of C. massanae in include Collyriclum within the family Troglotrematidae. The the bank vole Myodes glareolus from northeastern Poland. Troglotrematide proposed by Odhner (1914) was redefined by Dollfus (1939), leaving Collyriclum as the only genus within the * G. Kanarek ( ) Collyriclidae, a family placed within the superfamily Ornithological Station, Museum and Institute of Zoology, Polish Academy of Sciences, ul. Nadwiślańska 108, 80-680 Gdańsk, Poland Troglotrematoidea by Yamaguti (1971). In the latest classifica- e-mail: [email protected] tion based exclusively on morphological criteria, the Collyriclidae was included within the superfamily ś G. Zale ny Gorgoderoidea Loss, 1899 and consists of two monotypic gen- Institute of Biology, Wrocław University of Environmental and Life Sciences, ul. Kożuchowska 5b, 51-631 Wrocław, Poland era Collyriclum Kossack, 1911 and Collyricloides Vaucher, 1969 (Blair and Barton 2008); the latter erected to accommodate A. Czujkowska a digenean from rodents which lives in internal cysts and strong- Warsaw Zoological Garden, ul. Ratuszowa 1/3, ly resembles C. faba, but which possesses two suckers. Recently 03-461 Warszawa, Poland published analyses of variable 18S and 28S rDNA of C. faba J. Sitko have reassessed the relationships of Collyriclum and have placed Comenius Museum, Horní nám. 7, 750 11 Přerov, Czech Republic it unambiguously within the Microphalloidea (Heneberg and Literák 2013; Kanarek et al. 2014). P. D. Harris Natural History Museum, University of Oslo, P.O. Box 1172, The position of the genus Collyriclum is unquestionable, N-0562 Oslo, Norway but the status of Collyricloides is much more contentious. Parasitol Res Although similar to Collyriclum, Collyricloides massanae E. rubecula) following the manufacturer’s protocol (DNeasy was collected from intestinal cysts in the yellow-necked Blood and Tissue Kit, Qiagen, Germany). The 28S rDNA mouse Apodemus flavicollis from SW France and differs es- locus was amplified using primers: forward—dig12 (5′- pecially by the possession of a ventral sucker (Vaucher 1969), AAG CAT ATC ACT AAG CGG-3′) and reverse—1500R but also in several important details of the arrangement of the (5′-GCT ATC CTG AGG GAA ACT TCG-3′)(Tkachetal. genital- and excretory pores, the bursa cirri, the seminal recep- 2003). The ITS1-5.8S-ITS2 region was amplified with the tacle and the metraterm. Based on these differences, it seems following primers: NLF/NLR (5′-TTTGyACACACCGCC unlikely that Collyriclum and Collyricloides are closely relat- CGTCG-3′/5′-ATATGCTTAArTTCAGCGGGT-3′)(Vander ed, but to date, there has been no question as to their member- Auwera et al. 1994). PCR reactions were performed in a total ship of the Collyriclidae. We hypothesize that the resemblance volume of 25 μl containing 3 μl of genomic DNA, 10 mM between the two genera is superficial and convergent, and Tris–HCl, 50 mM KCl, 1.5 mM MgCl2, 200 μM of each results from similarities in cyst-dwelling habitat, rather than dNTP, 150 pmol of each primer, and 2 units of Taq polymer- being evidence of real phylogenetic affinity. In the present ase (EurX, Poland). The thermocycling profile was as follows: work, we evaluate the phylogenetic relationships of 95 °C/3 min—initial denaturation; 94 °C/30 s, 52 °C/30 s Collyriclum and Collyricloides within the family (28SrDNA) or 48 °C/30 s (ITS complex), 72 °C/90 s—40 cy- Collyriclidae, and present new data on the geographical dis- cles; 72 °C/7 min—final extension. tribution of Collyricloides. The amplification products were purified using QIAquick PCR purification Kit (Qiagen, Germany) and sequenced in both directions (Genomed S.A., Poland). The obtained se- Materials and methods quences were deposited in GenBank under accession numbers KP682451 and KP682452. Sampling protocols Alignment and phylogenetic analyses Material of digeneans from intestinal cysts were collected by dissection from a male Eurasian wren Troglodytes troglodytes, In order to elucidate any homologies with previously depos- admitted to the Rehabilitation Centre for Protected Birds, ited sequences in GenBank, we conducted a BLAST search Warsaw Zoological Garden which subsequently died. (www.ncbi.nih.gov/BLAST). This analysis showed that the Necropsy revealed five large cysts on the serosal surface of sequences of C. massanae were closest to representatives of the intestine; adult digeneans were isolated from the cysts, the superfamily Microphalloidea; thus, in the alignment, we washed in tap water, fixed, and stored in 70 % ethanol. use previously published sequences of Microphalloidea Additionally, five specimens of C. massanae were isolated (Table 1). Sequences were aligned using the MAFFT v.7 soft- from the cysts located on the intestine of adult male of ware (www.mafft.cbrc.jp/) with FFT-NS-1 option. The phylo- European robin Erithacus rubecula, found dead on 6 April genetic analysis was performed using Bayesian inference (BI) 2014 near Zahlinice, 15 km to south to Přerov, Kroměříž with the MrBayes ver. 2.01 software (Huelsenbeck and District, Moravia, Czech Republic. Specimens were isolated, Ronquist 2001); sequences of Fasciola hepatica processed as above and identified according to original de- (AY222244) was chosen as an outgroup. Bayesian inference scription (Vaucher 1969). Voucher specimens, stored in etha- was employed using the following nucleotide substitution pa- nol have been deposited in Polish Collection of Parasitic rameters: nst=6, rates=invgamma, that correspond to a gen- Helminths, Museum of Natural History, Wrocław, Poland, eral time reversible model (GTR) including estimates of the Coll. No. 144283 (specimens ex T. troglodytes) and 144284 proportion of invariant sites (I) and gamma distribution (G). (specimens ex E. rubecula). Further samples of C. massanae Posterior probabilities were approximated over 1,000,000 were derived from Myodes glareolus from Urwitałt forest, generations, log-likelihood scoters plotted and only the final northeastern Poland, where the parasite has been sporadically 75 % of trees were used to produce the consensus tree by found to occur. In this case, during fieldwork in 2008/2009, setting the ‘burnin’ parameter at 250,000. This number of two M. glareolus (out of c. 1000 examined over 10 years) generation was sufficient because the standard deviation were found infected with C. massanae.Inbothcases,upto dropped below 0.01 at the end of the run. seven parasites were located in cysts attached to the intestinal wall. Results DNA extraction, amplification and