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The presence of L. paradoxum and U. macrostomus among trematode specimens that could not be assigned to species on the basis of morphological characters was confirmed by molecular analysis. Trematodes L. paradoxum and U. macrostomus are typical species found in tits in Poland and other European countries. The DNA barcoding method used for genetic identification, based on analysis of variation in the COI gene sequence, proved successful in resolving doubts regarding the identification of trematodes found in Eurasian blue tit and Great tit as L. actitis and L. macrostomum. The results of the molecular analysis clearly confirmed the presence of two species of trematodes: L. paradoxum and U. macrostomus. L. paradoxum was shown to have more haplotypes and lower haplotype and nucleotide diversity than U. macrostomus. The differences were most likely due to the fact that the number (n = 21) of L. paradoxum trematodes identified was higher than the number of trematodes of the species U. macrostomus (n = 10). The genetic analyses included in this study were mainly meant to identify the species of trematodes that could not be classified on the basis of morphological characteristics. Therefore, confirmation and verification of the genetic diversity among the collected representatives of L. paradoxum and U. macrostomus require more extensive analyses based on a larger group of representatives of both species.
The tapeworm A. globata (Paruteniridae, Cyclophyllidea) was recorded in the present study for the first time in Poland. A. globata is widespread in many countries in Europe, including those along the migration route of tits, e.g., Germany, France, and Moldova [63]. Poland had previously been a gap on the map of Europe, which was filled owing to the research undertaken in this study. M. parinum is a cestode that is commonly recorded. It has previously been noted in Poland in Sturnus vulgaris [38]. The trematode L. paradoxum is a parasite of passerines and waders in Europe [64]. In Poland, this trematode has previously been recorded in Turdus merula, T. philomelos, C. caeruleus, and P. major, as well as in intermediate hosts, in practically all of Poland, including the migration route of tits from northeastern to northwestern Poland, together with the Baltic coast [38,49,50]. Outside of Poland, it has been recorded in Germany, Great Britain, Denmark, Finland, Moldova, Ukraine, Norway, Sweden, and the Czech Republic [45,48,63]. As indicated in the abovementioned works, trematodes of L. paradoxum are commonly present in Europe, including Poland. Thus, the identifications of L. paradoxum in migrating tits, both in the earlier studies and in our work, do not indicate that the migration of birds is the factor that influences infection of L. paradoxum but only shows that parasites are present in the birds during their migration.
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