The results reveal several new and important insights into the diversity of strigeid trematodes occurring in freshwater leeches in Central Europe and clearly emphasize the importance of this group of aquatic invertebrates in the circulation of trematodes in the aquatic environment. The observed prevalence of tetracotyle metacercariae detected in leeches (40.5%) was relatively high compared to other studies from central and eastern Europe: during investigations of parasites of species-rich and diverse communities of leeches in the eutrophic Drużno Lake (northern Poland) Dobrowolski  detected larvae of strigeid and cyathocotylid trematodes among 20% of analysed leeches, while in lakes of Kazakhastan Zhatkanbaeva  and Zhatkanbaeva and Akhmetova  detected tetracotyle metacercariae in 19.4% of the investigated leeches. On the other hand, studies from eastern Europe revealed the prevalence of tetracotyle metacercariae at much higher levels: in the Volga estuary Sudarikov et al.  found metacercariae in 60% (75 of 125) of analysed leeches, and Rajshite  detected strigeid metacercariae in 53% of leeches from the Volga and Niemen estuaries.
Tetracotyle metacercariae usually classified as Tetracotyle typica de Fillippi, 1855 have been widely recorded from snails and leeches in Europe, but their real taxonomic status has remained unknown for many years. Meanwhile, the adult form of T. typica sampled from snails has been identified as Cotylurus cornutus (Rudolphi, 1808) (for details see Sudarikov  and references therein). Szidat  reared adult trematodes from metacercariae of T. typica from the gonads of two species of leech (Erpobdella octoculata and Heamopis sanguisuga) and also identified them as C. cornutus. These results prove the possibility of the occurrence of tetracotyles of C. cornutus in two, clearly distinct groups of intermediate host, i.e. snails and leeches. The identification of T. typica from leeches as C. cornutus confirmed the results of Timon-David  and Dobrowolski . Using material collected from leeches, Szidat [45, 47] described for the first time another type of metacercariae, Tetracotyle gracilis, and identified the adult form of it as Apatemon gracilis (Rudolphi, 1819). During studies concerning the taxonomic composition of strigeid metacercariae from leeches of the Volga estuary Sudarikov et al.  confirmed the validity of T. gracilis as the adult form of A. gracilis but questioned the status of T. typica from leeches as C. cornutus. In their opinion, T. typica from leeches represents another species that is closely related to C. cornutus. Sudarikov et al.’s conclusions  were further confirmed by Vojtek et al. , who also stated that tetracotyle larvae of Cotylurus from leeches probably belong to a closely related, but different species of Cotylurus than tetracotyle larvae from snails that are commonly accepted in the contemporary literature as C. cornutus. However, these hypotheses were never confirmed and the issue of the identity and taxonomic position of Cotylurus metacercariae occurring in leeches and snails remained unresolved for years. According to the literature, the occurrence of tetracotyles of four species of Cotylurus has been detected in leeches: C. cornutus, C. hebraicus Dubois, 1934 C. strigeoides Dubois, 1958 and C. szidati Zazornova, 1991 [48, 49, 50, 51, 5]. Among these, tetracotyles of two species (C. cornutus, C. strigeoides) were also recorded from snail intermediate hosts [48, 5]. However, their real taxonomic position remains doubtful because of the subtle morphological differences between the tetracotyle forms of Cotylurus . Moreover, the taxonomic consequences of morphological variability observed among tetracotyles of Cotylurus have not been confirmed by molecular studies. For these reasons, recent knowledge concerning the morphology and taxonomic position of tetracotyles from snails and leeches should be considered as deeply insufficient and requires urgent verification using molecular studies based on well-determined reference materials from definitive hosts. Importantly, recent molecular studies have revealed unexpected high molecular diversity within Cotylurus [31, 14], including morphologically well-established species .
In the present study, metacercariae of Cotylurus detected in leeches were identified as two species, C. strigeoides and C. syrius, and this was confirmed by the parallel, comparative morphological and molecular analysis of adult specimens sampled from definitive avian hosts and GenBank sequences. Importantly, while leeches and snails are recognized as the second intermediate host for C. strigeoides [51, 5], the life cycle of C. syrius remains unknown. Our molecular identification of metacercariae detected in H. sanguisuga from Gdańsk Pomerania as C. syrius for the first time clearly indicates the route of transmission of this strigeid to the avian definitive hosts by leeches and emphasizes the importance of this invertebrate as a second intermediate host. Moreover, we consider the lack of tetracotyle forms of Cotylurus cornutus in leeches as really surprising. In Poland, this strigeid species is one of the most important elements ofthe trematode fauna of anseriform birds . The prevalence of C. cornutus in the population of mallard Anas platyrhynchos from Gdańsk Pomerania is about 20% (G. Kanarek, unpublished data), which could reflect the common occurrence of its invasive stages in the aquatic environment in the study area. In this context, the absence of metacercariae of C. cornutus in the helminth fauna of leeches suggests that the invasive form of this trematode species does not use leeches as intermediate hosts. This observation, along with other recently obtained results (identification of C. syrius and C. strigeoides in the leech helminth fauna), supports the hypothesis previously formulated by Sudarikov et al.  and Vojtek et al.  that tetracotyles of C. cornutus occur only in snail intermediate hosts, while the tetracotyle form detected in leeches represents different species of Cotylurus. Moreover, the real species composition and molecular diversity within the genus Cotylurus that utilises various invertebrate hosts (snails and leeches) remains unknown. Additionally, the presented results clearly indicate the separation of ecological niches and life cycles between some species of Cotylurus, with potential serious evolutionary consequences for a wide range of host–parasite relationships.
The obtained results clearly confirm the validity of the genus Cotylurus. All markers placed analysed members of this genus in one, well-supported clade with high diversity within the clade. However, the observed phylogenetic relationships within Cotylurus varied in relation to the analysed loci, which in our opinion, reflects the limited availability of fully reliable comparative sequences from related taxa in GenBank. Despite this, the sequenced specimens of C. strigeoides (both adults and tetracotyle) formed a well-supported separate clade that was particularly well defined in the phylogeny based on 28S rDNA loci (Fig. 2). In the analysis based on the ITS2 marker, isolates of C. strigeoides created a sister branch to a single isolate of C. gallinulae (JX978441) sampled from Aythya affinis in Mexico and provided by Hernández-Mena et al.  (Fig. 3). Heneberg et al.  suggested a high level of similarity between some lineages of C. cornutus (isolates from Anas crecca from the Czech Republic) and sequences C. gallinulae (JX978441), but our phylogeny, based on several new isolates and supplemented by GenBank data, did not confirm these assumptions. The current phylogeny based on ITS2 sequences (270 bp length) revealed only 0.4% difference (one nucleotide) between C. gallinulae (JX978441) from Mexico and recently obtained sequences of C. strigeoides (both adults and metacercariae), which strongly suggests that they are conspecific. Moreover, Locke et al.  suggested the identity of CO1 sequences of Cotylurus strigeoides sampled from Aythya collaris in Canada (MH581280-2) and C. gallinulae (JX977781) provided by Hernández-Mena et al. . Importantly, both JX978441 and recently sequenced adult specimens of C. strigeoides were sampled from anatid birds, which are recognized as the typical final hosts for this trematode species, while C. gallinulae is recognized as a typical parasite of Rallidae. According to Locke et al. , this suggests the possibility of incorrect determination of the material from Mexico. Another interesting question related to the position of C. gallinulae is the validity of recently sequenced specimens of Cotylurus hebraicus. This species was first described on the basis of trematodes collected from the Eurasian coot Fulica atra sampled in the territory of Syria and further placed as a subspecies within Cotylurus gallinulae as Cotylurus gallinulae hebraicus together with C. gallinulae gallinulae (Lutz, 1928) Dubois, 1937, C. gallinulae ban Yamaguti, 1939 and C. gallinulae vitellosus Lumsden et Zischke, 1963, which suggests their close affinity . All these taxa are recognized as typical parasites of coots, moorhens and rails, but with different geographical distributions. Some authors [54, 55] recognized C. hebraicus as a valid taxon with species rank, and this was confirmed in a recently obtained phylogeny, but the real taxonomic position and validity of other subspecies remain to be established. In our opinion, given their morphological similarity and narrow range of hosts, subspecies within C. gallinulae should be treated as a synonym of Cotylurus hebraicus. Confirmation of this hypothesis requires a detailed analysis of strigeid trematodes collected from typical avian final hosts (Rallidae) from a wide geographic distribution and comparison with sequenced specimens of C. hebraicus and other Cotylurus taxa.
Our results reveal the unclear taxonomic position and composite structure of C. syrius and C. cornutus, as reported previously by Heneberg et al. . According to these authors, trematode specimens morphologically identified as C. syrius and sampled from the typical host, the mute swan Cygnus olor, represent two distinct molecular lineages: one recognized as the typical C. syrius sensu stricto, and the other, according to Heneberg et al. , as a C. cornutus-like isolate due to its similarity to sequences obtained from adult specimens of C. cornutus sampled from Eurasian teal Anas crecca. Recently obtained sequences of the CO1 and ITS2 loci obtained from metacercariae sampled from H. sanguisuga from Gdańsk Pomerania and adult trematodes collected from a swan from Kraków are almost identical to sequences obtained by Heneberg et al.  from adult specimens of C. syrius sensu stricto (MF628093, MF628099 for ITS2; MF628057, MF628059 for CO1) (Fig. 3, 4). The identity of the abovementioned sequences was also confirmed using the 28S rDNA locus (Fig. 2). Additionally, sequences of the ITS2 fragment isolated from adult specimens of C. syrius sampled from Cy. olor from Lower Silesia were closely related to the sequence reported by Heneberg et al.  (MF628091) and described as C. cornutus-like isolates of C. syrius (Fig. 3). Phylogenetic reconstruction based on the CO1 locus and the GMYC analysis revealed a separate lineage clustering C. cornutus-like C. syrius with isolates of C. cornutus obtained from the typical definitive host, the mallard Anas platyrhynchos (Fig. 5). Sequences of the CO1 locus of C. cornutus obtained in the current study differed from sequences reported by Heneberg et al.  under the name C. cornutus (MF628064) and were almost identical to recently sequenced adult specimens and the tetracotyle of C. strigeoides (Fig. 4, 5). The obtained sequences supplemented with GenBank data clearly confirmed the existence of three distinct lineages within species morphologically identified as C. syrius: one lineage identified by Heneberg et al.  as C. syrius sensu stricto, recorded from the definitive host Cy. olor from the Czech Republic and Poland (Kraków) and in the tetracotyle from the leech H. sanguisuga from Gdańsk Pomerania, and two lineages identified by Heneberg et al.  as C. cornutus-like C. syrius. One of them grouped some specimens of C. syrius sampled from a typical definitive host, the swan Cy. olor, from the Czech Republic and Poland (Lower Silesia), and the second, collected from Cy. olor from the Czech Republic. The CO1 locus of the latter is almost identical to that of C. cornutus collected from the typical definitive host, the mallard A. platyrhynchos from Poland (Vistula Lagoon). In this context, swans in Central Europe can be parasitized by three (not two, as suggested previously by Heneberg et al. ), morphologically indistinguishable but molecularly different species of Cotylurus, classified as C. cornutus or C. syrius. In this regard, our results clearly reveal the urgent need to verify the validity and range of morphological criteria enabling the identification of adult specimens of some Cotylurus species.
Recently obtained data revealed the separate position of Cotylurs raabei within Cotylurus. For many years, this species was placed in the genus Cotylurus , Cotylurostrigea Sudarikov, 1961 [26, 54] or Strigea [25, 19]. Based on the results of morphological and cladistics analysis Zazornova and Sysoev  synonymised Cotylurostrigea with Cotylurus and placed C. raabei within the latter. In the phylogeny based on 28S rDNA and COI markers the position of this species is separate, forming a sister branch to the other Cotylurus species (Figs. 2 and 4), and C. raabei is clearly distinct, while in the phylogeny based on the ITS region this species is placed in a lineage between the recently sequenced C. hebraicus and isolates of cercarial Cotylurus sp. infecting Biomphalaria straminea in Brazil (MN179272 and MN179271) . The ITS2 sequences (270 bp) from C. raabei differed by 4.8% from C. hebraicus and by 5.2% from Cotylurus sp. from Brazil (MN179272 and MN179271). Given the inconsistent results obtained from the analysis of different loci, the taxonomic status of C. raabei remains unexplained and awaits further studies.
Another problem that is rarely mentioned in the contemporary literature is the identification, genetic variability and taxonomic position of metacercariae of Australapatemon from leeches. Due to their morphological similarity the metacercariae and adults of Australapatemon have for many years been erroneously confused with Apatemon e.g. . According to the literature, one of the features enabling fully reliable differentiation between the genera Australapatemon and Apatemon (other than the structure of excretory systems in cercariae) is the host of the invasive stages (fish in Apatemon, leeches in Australapatemon). However, this is not true in all cases. Negm-Eldin and Davies  revealed that metacercariae of Apatemon hypseleotris from Australia can develop in both leeches and fish. Regarding fact, that structure of cercariae of A. hypseleotris are typical for genus Australapatemon (14 flame cells), the taxonomic position of this species should be elucidated in further analysis . Another species of Apatemon with a doubtful taxonomic position and metacercariae in leeches is Apatemon jamesi . On the other hand, all species of Australapatemon with described life cycles utilise leeches as the hosts of invasive stages [58, 59, 19, 60, 10, 61, 6]. Unfortunately, studies concerning the identitification, taxonomic position and diversity of Australapatemon metacercariae occurring in leeches from various ecosystems are scarce.
Similar to the phylogenetic relationships within the genus Cotylurus discussed above, the phylogenetic relationships among Australapatemon varied with the loci analysed, which again, in our opinion, reflects the limited availability of fully reliable comparative sequences of related taxa in GenBank. Our data illustrate the inconsistent and confusing taxonomic status of sequenced tetracotyles of Australapatemon. Regarding the 28S rDNA fragment, the results revealed the greatest similarity to the sequences of Au. burti from Mexico (MF398342–99.9%, one nucleotide different), and Au. niewiadomski from New Zealand (KT334164, KT334165–99.4%, six nucleotides different) (Fig. 2). These results are in partial disagreement with sequences of ITS2 fragment recently sampled from metacercariae from leeches that showed 100% similarity with sequences described as Au. minor from Anas platyrhynchos from the Czech Republic (MF628095) and Au. burti from cercariae from Slovakia (KU950451) and the USA (KY570947). Moreover, to increase confusion, the sequences of ITS2 were also similar to those from a trematode identified as Au. mclaughlini (one nucleotide different compared to recently obtained isolates) and Au. burti from Mexico (two nucleotides different). The CO1 sequence analysis also gave inconclusive results: all sequences were placed in a separate clade, sister to Au. minor (MF628066) from Anas platyrhynchos from the Czech Republic (Fig. 4) and revealed high intraspecific diversity. Therefore, in the light of currently available molecular data in GenBank and our results, it is not possible to precisely define the taxonomic position of the recently sequenced tetracotyle of Australapatemon. The taxonomic position of the trematode identified as Australapatemon sp. collected from Anas strepera from Vistula Lagoon is also ambiguous: recently obtained sequences of 28S rDNA showed 100% homology with previously published sequences of Au. burti from Canada (KY207625)  and Mexico (MF398342) . Regarding the ITS2 fragment, our isolates presented the highest similarity with sequences of Australapatemon sp. (MK168687 and MK168688) from France . Based on the CO1 locus, an isolate of Australapatemon sp. from Anas strepera creates a separate branch to all other Australapatemon sequences available in GenBank, indicating its separate position (Fig. 4).
The phylogeny based on 28 rDNA and ITS2 strongly suggests unclear relationships between Au. burti and Au. minor. Australapatemon burti (Miller, 1923) was originally described from North America and often misreported as Apatemon gracilis [59, 63, 64, 65, 16], and has recently been widely recorded from the Holarctic (Central Europe) [66, 67, 22] and Neotropical regions [68, 69, 53, 6 and references therein], which strongly suggests the cosmopolitan distribution of this species. Au. minor Yamaguti, 1933 is recognized as a typical parasite of Anseriformes in the Palearctic region [18, 70]. Therefore, the sympatric occurrence of these two species (Au. burti and Au. minor) in the territory of Central Europe would not be surprising. Moreover, the study area in Gdańsk Pomerania (near the Gulf of Gdańsk and Vistula Lagoon) is recognized as a well-known refuge of water and wetland birds. In addition to the rich and diversified fauna of breeding birds, these places are an important resting place for birds migrating mainly from the north and north-east , which creates the possibility of bringing some species of helminths by migrating birds from nesting grounds located e.g., on the Scandinavian Peninsula or Siberia. For this reason, helminth taxa unusual for this geographical region could appear, especially in avian definitive hosts during migration. Such conditions require extreme caution in the interpretation of molecular data. Unfortunately, while fragments of 28S rDNA clearly revealed the similarity of recently obtained isolates from tetracotyle and adult Austalapatemon sp. with sequences of A. burti, sequences of ITS2 isolates from tetracotyle showed 100% homology with sequences described as Au. minor and Au. burti. The main underlying problem is that the only sequences of ITS2 and CO1 fragments of Au. minor available in GenBank and provided by Heneberg et al.  are rather short (270 bp for ITS2 and 295 bp for COI), which forced the shortening of our sequences and precluded the full comparison of these sequences with other data (based on full-length fragments). In phylogenetic analysis fragments of this limited length have limited significance and reduce the reliability of any conclusions.
Our results revealed the discursive and inconsistent status of the genera Apatemon and Australapatemon. In the phylogenies based on 28S rDNA and ITS2, all analysed Australapatemon sequences were placed in one, well-supported clade, separate from other Strigeidae genera (Cotylurus and Apatemon), with the exception of sequences of Apatemon fuligulae, which were located between Australapatemon (Fig. 2, 3). In the phylogeny based on COI fragments, our sequences of Ap. fuligulae were almost identical to sequences of Ap. fuligulae submitted by Heneberg et al.  (MF628055), which confirmed the valid determination of this species. Importantly, both isolates were placed in a branch with other Apatemon species. The different generic position of Ap. fuligulae obtained in phylogenies based on various other loci may indicate the inappropriate current status of this species. These suspicions were confirmed by the phylogeny based on 28S rDNA fragments: sequences of Ap. fuligulae were similar to Canadian sequences of Australapatemon sp. (MF124270, three nucleotides different) provided by Gordy et al.  and isolated from an adult trematode specimen collected from the northern pintail Anas acuta and cercariae emerging from Stagnicola elodes. On the other hand, Yamaguti  detected the metacercariae of Ap. fuligulae (Tetracotyle fuligulae) in the skin and musculature of fish (Parasilurus asotus, Pseudobagrus aurantiacus) from Lake Biwa. Therefore, the taxonomic position of Ap. fuligulae should be considered as doubtful. Establishing its position will require further studies concerning both the genetic variability within this species and a detailed analysis of the morphology of cercariae and adults and the life cycle of Ap. fuligulae.
In conclusion, in our opinion, the unclear and inconsistent status of Apatemon and Australapatemon, visible especially using the CO1 and ITS markers, results mainly from the invalid determination of sequenced adult and larval trematodes: from a morphological point of view, as mentioned in the Introduction, the two genera are quite similar and often misidentified in the literature. Erroneous determination of sequenced adult and larval stages of both Apatemon and Australapatemon result in the erroneous description of the sequences deposited in GenBank, which increases the chaos in phylogenies based on them. Based on our own experience concerning the morphology of these genera and their ecology, as well as the literature, which show key differences between the life cycles of Apatemon and Austalapatemon, we believe that these genera are valid and distinct, as confirmed by the 28S rDNA sequences. However, full confirmation of this assertion requires a meticulous review of all sequences of Apatemon and Austalapatemon deposited in GenBank, which is not possible without detailed resolution of the life cycles of particular species within these genera.