The phylogenetic position of the enigmatic Balkan Aulopyge huegelii (Teleostei: Cyprinidae) from the perspective of host-specific Dactylogyrus parasites (Monogenea), with a description of Dactylogyrus omenti n. sp.

Background The host specificity of fish parasites is considered a useful parasite characteristic with respect to understanding the biogeography of their fish hosts. Dactylogyrus Diesing, 1850 (Monogenea) includes common parasites of cyprinids exhibiting different degrees of host specificity, i.e. from strict specialism to generalism. The phylogenetic relationships and historical dispersions of several cyprinid lineages, including Aulopyge huegelii Heckel, 1843, are still unclear. Therefore, the aims of our study were to investigate (i) the Dactylogyrus spp. parasites of A. huegelii, and (ii) the phylogenetic relationships of Dactylogyrus spp. parasitizing A. huegelii as a possible tool for understanding the phylogenetic position of this fish species within the Cyprininae lineage. Results Two species of Dactylogyrus, D. vastator Nybelin, 1924 and D. omenti n. sp., were collected from 14 specimens of A. huegelii from the Šujica River (Bosnia and Herzegovina). While D. vastator is a typical species parasitizing Carassius spp. and Cyprinus carpio L, D. omenti n. sp. is, according to phylogenetic reconstruction, closely related to Dactylogyrus species infecting European species of Barbus and Luciobarbus. The genetic distance revealed that the sequence for D. vastator from A. huegelii is identical with that for D. vastator from Barbus plebejus Bonaparte, 1839 (Italy) and Carassius gibelio (Bloch, 1782) (Croatia). Dactylogyrus omenti n. sp. was described as a species new to science. Conclusions Our findings support the phylogenetic position of A. huegelii within the Cyprininae lineage and suggest that A. huegelii is phylogenetically closely related to Barbus and Luciobarbus species. The morphological similarity between D. omenti n. sp. and Dactylogyrus species of Middle Eastern Barbus suggests historical contact between cyprinid species recently living in allopatry and the possible diversification of A. huegelii from a common ancestor in this area. On other hand, the genetic similarity between D. vastator ex A. huegelii and D. vastator ex C. gibelio collected in Balkan Peninsula suggests that A. huegelii was secondarily parasitized by D. vastator, originating from C. gibelio after introduction of this fish species from Asia to Europe.


Background
Parasites and their hosts are usually closely associated due to their coevolution, realized by reciprocal genetic adaptations between these interacting species. In evolutionary time, this leads to a selection for improvements in host-parasite recognition mechanisms [1]. The high degree of host specificity among parasites (generally, a parasite species is restricted to a single host species), reflecting parasite specialization, may arise from such coevolutionary interactions [2][3][4] In the case of high host specificity, the phylogeny of host-specific parasites may even follow the phylogeny and historical biogeography of their hosts as a result of co-speciation [5,6]. However, parasite diversification can also be driven by host specialization following host switching resulting from strong ecological association, as was shown for monogeneans of marine fish [7]. The host specificity of freshwater fish parasites appears to be a useful characteristic in terms of understanding the biogeography of freshwater fishes (e.g. [8][9][10][11]). Basic host specificity is commonly expressed by the number of host species (also termed host range). However, other aspects, like the ecological performance of the parasite, the phylogenetic affinities of hosts, and the biogeographical distribution of the parasite, are important when expressing host specificity [12].
Gill ectoparasites of the genus Dactylogyrus Diesing, 1850 generally exhibit a high degree of host specificity and a high species diversity arising from the multitude of cyprinid fish species, which are common hosts of these parasite species [13]. Šimková et al. [14] defined several levels of host specificity for Dactylogyrus using an index of host specificity, expressed as the inverted value of the index of non-specificity proposed by Desdevises et al. [7]. Five Dactylogyrus groups were defined ranging from strict specialists, which occur on a single host species, to true generalists, which parasitize different, phylogenetically unrelated cyprinid host species. These host-specific parasites have a direct life-cycle, in which the larval stage (oncomiracidium) actively searches for suitable host species, using chemical cues for host recognition [15]. Therefore, among monogeneans, a high degree of adaptation to their host resource is required [16][17][18][19]. Several studies documented microhabitat restriction (i.e. preferred niche measured by specific gill positions) in Dactylogyrus species [20][21][22][23][24]. Since different parts of gills offer different types of substrate, niche preference is associated with a specific type and shape of attachment organ (haptor) in parasites assigned to Dactylogyrus [9,21,23]. Šimková et al. [23] also revealed that there is morphological adaptation of the haptor in species that specifically parasitize phylogenetically related hosts, such as Dactylogyrus species parasitizing Cyprinus carpio L. and Carassius auratus L. of the subfamily Cyprininae. The phylogeny of highly hostspecific Dactylogyrus species reflects the biogeography and evolutionary history of their cyprinid hosts [25]. Besides some accidental infections of unsuitable hosts, the sharing of Dactylogyrus species among evolutionary divergent cyprinid species living in sympatry is rare [23].
The cyprinid fauna of the Balkan Peninsula is extremely rich in endemic species [26]. According to Oikonomou et al. [27], the Balkan freshwater fish fauna represents 59% of all known cyprinid species. The ancient Dessaretes lake system played an important role in cyprinid speciation, which originated during the Pleistocene and is considered as a hotspot of endemic freshwater biodiversity [28][29][30][31][32]. Presently, all the great lakes in the Balkan Peninsula, the Ohrid, Prespa, Mikri Prespa and Maliq lakes (the latter one was drained after World War II), are parts of this system. Albrecht & Wilke [30] also theorized that during the Miocene and Pliocene eras the whole Dessaretes basin was filled with water and that all lakes were connected. After the closing of the Korca Depression and connections between the Dessaretes and the Paratethys, the water level decreased and fragmentation of the populations triggered allopatric speciation, which led to rich freshwater fish diversity. Zardoya et al. [33] investigated the geographical origin of Balkan endemic cyprinids. They suggested that cyprinid fauna colonized the Balkan Peninsula during two different time periods. The first wave occurred during the Miocene and the second during the Plio-Pleistocene via river captures. The phylogenetic relationships among Balkan cyprinid taxa and their biogeographical histories have been actively studied over the last 25 years (e.g. [34][35][36][37][38][39][40]). Studying host-specific parasites, such as Dactylogyrus, may represent an additional tool for investigation and may shed more light on both the historical contacts between cyprinid hosts and their phylogeography.
The Dalmatian barbelgudgeon (Aulopyge huegelii Heckel, 1843), the only representative of the monotypic genus Aulopyge, is one of the many endangered cyprinid species of the Balkan Peninsula. Its distribution is limited to the Dinaric karst rivers and lakes of Croatia and Bosnia and Herzegovina [41][42][43]. Although previously quite abundant, in recent years A. huegelii populations have been declining [44]. Tsigenopoulos & Berrebi [43] considered the ancestor of A. huegelii as the first migration wave of cyprinids to the Mediterranean region, which found refuge in Dalmatia. According to the molecular clock, they estimated that European Barbus and A. huegelii diverged during the middle Miocene, which concurs with the first wave colonizing Balkan Peninsula [33]. On the basis of mitochondrial cytochrome b sequence data, Tsingenopoulos et al. [45] suggested that A. huegelii is the sister clade to the clade including Barbus + Luciobarbus lineages. However, Yang et al. [46] showed that Aulopyge is the sister taxon to the the European Barbus lineage, well separated from the Luciobarbus lineage, and, according to Wang et al. [47], the European Barbus (sensu stricto) lineage and A. huegelii share a common ancestor (originating in the Qinghai-Tibetan Plateau region about 19.4-7.8 Mya) with the species of the Asian genera Schizothorax and Cyprinion.
Until now, only a very few endemic cyprinid species from the Balkan Peninsula have been investigated for parasites [48][49][50][51][52][53][54]. As previously shown by Šimková et al. [25], phylogenetic relationships between Dactylogyrus lineages can reflect cyprinid phylogeny. Thus, we hypothesized that the phylogenetic relationships between host-specific Dactylogyrus species of A. huegelii and those parasitizing other closely related cyprinid species will support the phylogenetic position of this monotypic cyprinid genus. Therefore, the aims of our study were (i) to investigate the Dactylogyrus fauna of endemic A. huegelii, and (ii) to investigate the phylogenetic relationships between Dactylogyrus species parasitizing A. huegelii and those parasitizing species of the Cyprininae distributed in Europe, i.e. Barbus spp., Carassius spp. and C. carpio (the last two originating from Asia and widely distributed throughout the whole of Europe). As a result, we described a new species of Dactylogyrus collected from endemic A. huegelii.

Sampling and species identification
A total of 14 specimens of Aulopyge huegelii from the Šujica River, Bosnia and Herzegovina, were sampled in July 2015. Fish were dissected using standard methods [55]. Dactylogyrus specimens were collected from host gills, fins, head surfaces, and oral and nasal cavities, mounted on slides and covered with a mixture of glycerine and ammonium picrate (GAP [56]) for further identification. The identification of monogeneans was performed using Gussev [57] on the basis of the size and shape of the hard parts of the attachment organ, the haptor, and the reproductive organs which represent species-specific morphological characters. Identification to species level was performed using an Olympus BX51 microscope equipped with phase contrast optics. Several Dactylogyrus specimens were bisected; one half of the body (usually the half with the reproductive organs) was mounted on a slide for species identification, the other was individually preserved in 96% ethanol for DNA extraction. Basic epidemiological data, i.e. prevalence, mean abundance, minimum and maximum intensity of infection, were calculated for each species according to Bush et al. [58]. Prevalence, as the percentage of fish infected by a given parasite species, and mean abundance, as the mean number of parasite specimens per individual host taking into account both infected and uninfected hosts, were calculated.

Morphometric data
Morphometric measurements of Dactylogyrus spp. specimens (modified according to Gussev [57]) were taken using Digital Image Analysis (Stream Motion). All measurements of morphometric characters are in micrometres and are presented as the range followed by the mean and the number of measured specimens (n) in parentheses. The numbering of marginal hook pairs for Dactylogyrus follows the recommendations by Mizzele [59]. After measuring morphometric characters, the specimens were removed from GAP and remounted in Canada balsam, according to Ergens [60], and deposited as type-specimens in the Helminthological Collection of the Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, in České Budějovice (IPCAS).

DNA extraction, amplification and sequencing
Parasites were removed from storage ethanol and dried by means of a vacuum centrifuge. DNA extraction was performed using a standard protocol (DNeasy Blood & Tissue Kit, Qiagen, Hilden, Germany). Partial 18S rDNA and entire ITS1 regions were amplified using primers S1 (5′-ATT CCG ATA ACG AAC GAG ACT-3′) and IR8 (5′-GCT AGC TGC GTT CTT CAT CGA-3′) [61], which anneal to the 18S and 5.8S rDNA regions, respectively. Each amplification reaction for partial the 18S rDNA and ITS1 regions was performed in a final volume of 15 μl, containing 0.3 μl of Taq polymerase, 1.5 μl buffer, 0.9 μl MgCl 2 , 0.3 μl of dNTPs, 1.5 μl of each primer and 2.5 μl of pure DNA (20 ng/μl). PCR was carried out using the following steps: 2 min at 94°C, followed by 40 cycles of 1 min at 94°C , 1 min at 53°C and 90 s at 72°C, and 10 min of final elongation at 72°C. Partial 28S rDNA was amplified using the forward primer C1 (5′-ACC CGC TGA ATT TAA GCA-3′) and the reverse primer D2 (5′-TGG TCC GTG TTT CAA GAC-3′) [62]. PCR followed the protocol included in Šimková et al. [14]. PCR products were checked on 1.5% agarose gels, purified by using an ExoSAP-IT kit (Ecoli, Bratislava, Slovakia), following the manufacturer's protocol, and sequenced directly using the PCR primers and BigDye Terminator Cycle sequencing kit (Applied Biosystems, Pardubice, Czech Republic). Sequencing was carried out using an ABI 3130 Genetic Analyzer (Applied Biosystems). The newly generated sequences were deposited in the GenBank database and molecular vouchers (hologenophores, paragenophores [63]) were deposited in the Helminthological Collection of the Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, in České Budějovice (IPCAS).

Phylogenetic analyses
DNA sequences were aligned using fast Fourier transform in MAFFT [64]. To match the lengths of the newly obtained sequences to the sequences obtained from GenBank, they were optimized manually. A test of homogeneity to examine the congruence of two datasets (partial 18S with the ITS1 region vs 28S rDNA) was performed in PAUP* 4b10 [65]. Since the difference was not statistically significant (P = 0.737), the concatenated data were used for further phylogenetic analyses. were included in phylogenetic analyses due to the proposed evolutionary proximity of the host species. The final tree was rooted using Dactylogyrus species of C. gibelio and C. carpio as the outgroup taxa, following Šimková et al. [23].
To analyze the genetic distances between the specimens of D. vastator from different host species, sequences of partial 18S rDNA and complete ITS1 available for D. vastator were obtained from GenBank. The uncorrected p-distances between D. vastator collected from 5 different host species from 7 localities were calculated using MEGA6 [66].
Gaps and ambiguously aligned regions were removed from the alignment using GBlocks v. 0.91 [67]. The most appropriate DNA evolution model was determined using the Akaike information criterion (AIC) in JModelTest 2.1.10 [68,69]. Phylogenetic trees were inferred by Bayesian inference (BI) and Maximum Likelihood (ML) analyses using MrBayes 3.2 [70] and PhyML 3.0 [71], respectively. The search for the best ML tree was performed using NNI (nearest neighbour interchange) and SPR (subtree pruning and regrafting) branch swapping algorithms with six substitution categories. The clade support for ML was assessed by 1000 bootstrap pseudoreplicates. Bayesian inference trees were constructed using the MC 3 algorithm with two parallel runs containing one cold and three hot chains. The analysis ran for 10 7 generations and tree topologies were sampled every 100 generations. The first 25% of all saved trees were discarded as relative 'burn-in' periods according to standard deviation split frequencies (< 0.01). Posterior probabilities were calculated as the frequency of samples recovering any particular clade.

Parasites of A. huegelii
All 14 dissected fish specimens were infected with monogenean parasites. Dactylogyrus spp. reached 93% prevalence in A. huegelii and represented two species. The first was Dactylogyrus vastator, a common parasite of Carassius spp. and C. carpio, and which also accidentaly infects some other fish species ( [13], M. Benovics, unpublished data). Morphological identification confirmed that specimens of D. vastator from A. huegelii possess the same morphology of the hard parts of the haptor and reproductive organs (i.e. the shape was identical and the size of these parts was within the range of sizes included in original description of D. vastator). The second species is here described as Dactylogyrus omenti n. sp., which was not found on other endemic Barbus species, or any other cyprinids collected in the Balkan Peninsula, and is most likely specific to A. huegelii. Both Dactylogyrus species differed in their epidemiological characteristics ( Table 1). The prevalence of D. omenti n. sp. was significantly higher than that of D. vastator (Fisher's exact test, P = 0.006, df = 1). The abundance of D. omenti n. sp. was higher than that of D. vastator (Mann-Whitney test, U (14) = 15.00, Z = 3.79, P < 0.001).

Phylogenetic position of Dactylogyrus spp. parasitizing A. huegelii
A final concatenated sequence alignment was constructed using 1625 unambiguously aligned nucleotide positions. GTR + I + G was selected as the optimal evolution model. ML and BI analyses provided phylogenetic trees with similar topologies. The BI tree is presented in Fig. 1, where bootstrap values resulting from ML analysis and posterior probabilities resulting from BI analysis are included. Collection localities and GenBank accession numbers of all newly generated sequences used in the phylogenetic reconstructions are provided in Table 2.
The resulting tree for Dactylogyrus spp. supports the close phylogenetic relationship of A. huegelii to endemic Mediterranean Barbus and Luciobarbus species and to the widely distributed European Barbus barbus, as previously shown by molecular phylogenetic studies of cyprinid fishes [43,45,47,72] Luciobarbus. All these species exhibit a similar shape of the haptoral hard parts, especially in having a cross-shaped connective ventral bar with 5 marginal extremities, but differ between clades in the shape of the MCO. The last supported grouping (group C in Fig. 1     GenBank accession numbers included Genetic distances were calculated using the sequences of partial 18S rDNA and ITS1 (see Table 2 for accession numbers for D. vastator sequences generated in this study)   [48,79]. However, D. omenti n. sp. differs from these species by the size of its haptoral hard parts, which are smaller (comparative morphometric data are provided in Table 4). In general, the configuration of hard haptoral elements and the shape of the ventral bars also resembles Dactylogyrus spp. from Moroccan Luciobarbus spp. described by el Gharbi et al. [80]. The MCO of D. omenti n. sp. most closely resembles the MCO of D. deziensioides, due to the presence of the colonshaped process of the accessory piece encircling the copulatory tube. However, the copulatory tube of D. deziensioides is massive and short, in contrast with the delicate and long copulatory tube of D. omenti n. sp. In the original description of D. affinis, Bychowsky [81] pointed out the poor visibility of the end of copulatory tube, because of a saucer-shaped broadening of the accessory piece. This observation corresponds with the poor visibility of the medial part of the copulatory tube of D. omenti n. sp., on account of the shield-like broadening. Nevertheless, the colonshaped process of the accessory piece is missing in the original drawing of D. affinis. The elongated twisted vagina of D. affinis markedly resembles the shape of the vagina of D. omenti n. sp. In regards to D. crivellius, D. omenti n. sp. differs in having a longer copulatory tube, larger colon-shaped part of the accesory piece and a thinner and longer vagina. sp. was recognized only from A. huegelii in this study, and therefore it is likely specific for this cyprinid species.

Discussion
Dactylogyrus vastator, the parasite species with a large body size, has been widely reported from wild and farmed populations of C. carpio and Carassius spp., both of which belong to the subfamily Cyprininae (e.g. [52,[83][84][85][86]). In addition, the accidental infection of D. vastator was also found on some other cyprinid species (especially on Barbus [13], M. Benovics, unpublished data). Our study revealed a moderate prevalence of D. vastator on A. huegelii, which indicates that the infection of D. vastator on this endemic cyprinid species is not an accident. However, the low parasite infrapopulation size may indicate that this host is probably not suitable for maintaining parasite populations. Cyprinus carpio and C. gibelio may harbour up to nine different Dactylogyrus species [25,87,88]. The presence of only D. vastator on A. huegelii from this wide range of Dactylogyrus species could indicate: (i) the absence of other Dactylogyrus spp. on C. carpio and Carassius species potentially living in sympatry with A. huegelii; (ii) strict host specificity among other Dactylogyrus spp. of C. carpio and C. gibelio resulting from reciprocal coadaptation; or (iii) different morphologies of gill filaments providing microhabitats suitable for some Dactylogyrus species (i.e. large species such as D. vastator or D. extensus), but unsuitable for others (i.e. small species such as D. achmerowi Gussev, 1955, D. falciformis Akhmerov, 1952 or D. minutus Kulwiec, 1927). To test these hypotheses, further investigation of parasite communities on C. carpio and Carassius spp. potentially living in sympatry with A. huegelli and analyses of the niche preferences of Dactylogyrus parasites (i.e. the preferred positions on fish gills) are necessary. Dactylogyrus vastator usually infects small fingerlings, where overpopulation may result in the mortality of the host. According to Uspenskaya [89], 40 specimens of D. vastator could possibly cause the death of a fish with a body length of 2 cm. This is not the case with A. huegelii, where very low abundance was found, i.e. only a single specimen of D. vastator per individual fish, suggesting that mortality of this host is unlikely. This low abundance is conflicting with optimal conditions for the development of this parasite species [90,91], because high population growth and consequently a high intensity of infection on the part of D. vastator are expected in southern regions, which have high water temperatures in summer. Possible explanations could be that the mobility of D. vastator larvae is restricted by different suboptimal environmental factors, resulting from the habitat preference of A. huegelii; that is, finding new hosts in these conditions may be more difficult. Alternatively, this species could be competitively excluded by higher populations of the second host-specific species parasitizing A. huegelii, Dactylogyrus omenti n. sp. [79,92], which, in our study, was the most abundant Dactylogyrus species on A. huegelli.
We hypothesized that Dactylogyrus species are a good indicator of evolutionary relationships between cyprinid host species. Despite the low abundance of D. vastator on A. huegelii, this record supports the phylogenetic relationships of A. huegelii to species of the Cyprininae originating from Asia and probably introduced into Europe, i.e. C. carpio and Carassius spp. This parasite species was also found in very low abundance (1 specimen per fish and a prevalence of 20%) on Barbus plebejus during our field study in Italy. Aulopyge huegelii possibly offers a similar type of substrate, which, in the case of Dactylogyrus spp., is gill filaments, and, therefore, common Dactylogyrus spp. parasitizing C. carpio and Carassius species [83,85,88,93] can also develop and inhabit closely phylogenetically related species such as A. huegelii and some Barbus species. This  [91] may support the finding of Shamsi et al. [88] indicating that the transmission of D. anchoratus from common carp to Barbus sharpeyi, an important native fish species, takes place despite the high host specificity of many Dactylogyrus species. Šimková et al. [25] proposed that the phylogeny of Dactylogyrus reflects, at least partially, the phylogeny of their cyprinid host species (depending more or less on the level of host specificity of particular species). According to Kohlmann et al. [94], European and Asian cyprinids share a common ancestor from central Eurasia. While C. carpio is widely distributed in the Eurasian region, species of the C. auratus complex are native to eastern Asia and were only recently imported into Europe and other continents [26,95]. There are no paleontological records of the C. auratus complex in Europe before the Pleistocene [95].  [43]. Unfortunately, the lack of molecular data for D. affinis and D. deziensioides makes further examination of evolutionary connections currently impossible. With the shape of its haptoral hard elements, especially its typical cross-shaped ventral bar with five extremities, D. omenti n. sp. resembles Dactylogyrus of Greek and Moroccan Luciobarbus (see [80] for their morphology) and also D. carpathicus and D. crivellius from widely distributed Barbus species [48,82]. It was suggested that the shape of the haptoral hard parts appears to be more suitable for resolving phylogenetic relationships between lineages of a given monogenean genus, while the shape of the reproductive organs is more suitable for identification at the species level because of its faster evolutionary change [23,[96][97][98][99]. This may indicate that D. omenti n. sp. is evolutionarily closer to the earlier mentioned species than to other Dactylogyrus of Barbus, possessing a different type of ventral bar. Nevertheless, our results showed that four Dactylogyrus spp. with a crossshaped ventral bar with 5 extremities, i.e. D. crivellius, D. carpathicus, Dactylogyrus sp. 1 and Dactylogyrus sp. 2 (clade B in Fig. 1), formed a well supported (PP = 0.95, BS = 64) monophyletic group to the exclusion of D. omenti n. sp. The unexpected phylogenetic position of D. omenti n. sp. indicates that using only the shape of the haptor as a marker for solving phylogenetic relationships in monogenean species with rapid diversification is not advisable and that the shape of the reproductive organs should be taken into account. However, the phylogenetic relationships between other Dactylogyrus species included in our phylogenetic reconstruction follow haptor morphology, specifically the shape of the connective bars and hooks. This is true of the monophyletic group of D. balkanicus and D. dyki (group C), which possess a similar shape of hard parts of attachment organ (anchors, connective bars and marginal hooks) [48], though the two species vary in the dimensions of their haptoral hard parts [79]. Additionally, the copulatory organ of both species is similar. The fast development of variations in reproduction organs is considered as a mechanism for avoiding hybridization in the case of multiple congeneric monogenean species living in overlapping microhabitats [100]. This is also true for Dactylogyrus species parasitizing Barbus. Of a possible seven Dactylogyrus spp., Barbus and Luciobarbus species usually harbour only Dactylogyrus species with copulatory organs of a markedly different shape, representing different phylogenetic lineages ( [80,92,101]. For instance, as is shown in the present study, B. prespensis hosts four species with differently shaped copulatory organs, D. balkanicus, D. crivellius, D. dyki and D. prespensis, representing three different phylogenetic lineages (see Table 2 and Fig. 1). Also the morphologically similar and phylogenetically close species, such as D. dyki and D. balkanicus, were not present on the same host species in one population.

Conclusions
Dactylogylrus omenti n. sp. was recognized as a potentially strict specialist of A. huegelii. Concluding from the expected high degree of host specificity of Dactylogyrus parasites and presence of D. vastator, a typical parasite of C. carpio and Carassius spp., on A. huegelii, or the phylogenetic position of D. omenti n. sp., the A. huegelii is a taxon closely related to European Barbus and Luciobarbus and to the Cyprininae of Asian origin. Regarding hard morphological characters, D. omenti n. sp. resembles species of Dactylogyrus parasitizing species of Barbus and Luciobarbus from the Middle East and Kazakhstan. Similarities in the shape of hard parts may suggest the origin of D. omenti n. sp. in this region and also an evolutionary proximity of endemic Cyprininae from the Middle East and Kazakhstan to A. huegelii. The genetic distances between D. vastator collected from different host species revealed that D. vastator in A. huegelii is identical with D. vastator of Balkan C. gibelio and closer to the central European C. carpio rather than to C. auratus complex. These results are indicating recent host switch of D. vastator between different hosts in Europe. The phylogenetic reconstruction of Dactylogyrus species parasitizing different endemic Barbus spp. and Luciobarbus spp. in the Balkan Peninsula and widely distributed European Barbus spp. revealed that, despite the generally accepted view that the morphology of the attachment organ is the best tool for resolving phylogenetic relations (based on morphological characters only) between Dactylogyrus species, the shape and size of the copulatory organs of rapidly evolving monogeneans have to be taken into consideration. But most importantly, only the combination of both morphological characters together with molecular data should be used for resolving the phylogeny and detection of potentially hidden diversity.