Uncovering the diversity of monogeneans (Platyhelminthes) on endemic cypriniform fishes of the Balkan Peninsula: new species of Dactylogyrus and comments on their phylogeny and host-parasite associations in a biogeographic context

Seven new species of Dactylogyrus Diesing, 1850 (Dactylogyridae) are described from the gills of seven endemic species of cyprinoids (Cyprinidae, Leuciscidae) inhabiting the Balkan Peninsula: Dactylogyrus romuli n. sp. from Luciobarbus albanicus (Greece), Dactylogyrus remi n. sp. from Luciobarbus graecus (Greece), Dactylogyrus recisus n. sp. from Pachychilon macedonicum (Greece), Dactylogyrus octopus n. sp. from Tropidophoxinellus spartiaticus (Greece), Dactylogyrus vukicae n. sp. from Delminichthys adspersus (Bosnia and Herzegovina), Dactylogyrus leptus n. sp. from Chondrostoma knerii (Bosnia and Herzegovina), and Dactylogyrus sandai n. sp. from Telestes karsticus (Croatia). To delineate species boundaries, we used an integrative taxonomic approach combining morphological and genetic data. With these tools, we illustrate that some species of monogeneans considered as cryptic might be designated as pseudocryptic (morphologically similar, not easily differentiated) after a posteriori detailed morphological examination, as happened with D. romuli n. sp. and D. remi n. sp. Thus, for accurate species characterization, it is particularly important to acquire both morphological and molecular data from the same individual specimens, ideally along with illustrations of taxonomically important structures directly taken from hologenophores. Using phylogenetic reconstruction, we investigated the phylogenetic position of newly described Dactylogyrus species within Dactylogyrus species from Balkan cyprinoids with regard to morphological characteristics, host range, and geographical distribution.


Introduction
The Mediterranean basin of the Balkan Peninsula is recognised as a global biodiversity hotspot [49].The fauna of this region is characterized by the exceptionally high diversity and endemism of freshwater fishes, especially Cypriniformes [40,50].In the last few decades, a number of investigations using molecular data have been devoted to investigate the relationships among Balkan cypriniform taxa and to infer their biogeographical histories [8,9,52,87].However, only a few studies [3,4,77] have focused on investigating the distribution and phylogeny of their host-specific parasites, such as monogeneans, which may reflect the evolutionary history of their associated cypriniform hosts.
Parasites with a high degree of host-specificity are generally expected to show close co-evolutionary relationships with their hosts [39,55].As monogeneans have a direct life-cycle and exhibit relatively high host-specificity, they represent powerful model organisms to search for a link between host and parasite diversification in ecological and evolutionary contexts [56].Among monogeneans, Dactylogyrus Diesing, 1850 (Dactylogyridae) is the most speciose genus, with more than 900 nominal species described mostly from the gills of cyprinoids [28].Morphological characters of the sclerites of the attachment organ (i.e., haptor) as well as of the copulatory organs (i.e., male copulatory organ and vagina) are commonly used for monogenean species identification [58].In recent years, however, a number of previously unrecognized (morphologically cryptic) parasite species have been revealed using molecular markers [57].Within monogeneans, a molecular approach has been applied especially to delimitate and describe "morphologically simple" species of Gyrodactylus von Nordmann, 1832 (Gyrodactylidae) (e.g.[42,61,92]), whose taxonomy depends primarily on the shape of the haptoral structures, especially the marginal hooks [27,45,70].Although Dactylogyrus species have many accessible morphological characters (i.e.those of the haptor and the reproductive system) compared to Gyrodactylus spp., cryptic species within this genus have also been detected.Recently, Rahmouni et al. [59]  In terms of host specificity, the majority of Dactylogyrus species are highly host-specific.However, the host specificity of Dactylogyrus species may range from strict specialism (i.e.species restricted to a single host species) and intermediate specialism (i.e.species restricted to a single host genus) to intermediate generalism (i.e.species parasitizing phylogenetically related non-congeneric hosts) and true generalism (i.e.species parasitizing phylogenetically unrelated hosts) [76].On the basis of the list of Dactylogyrus species identified by Benovics et al. [4] on endemic cyprinoids from the Balkans, we can calculate that each species parasitizes a mean number of 1.2 host genera; 94% of the species are reported from a single genus.As many Dactylogyrus species are restricted to a single host species, members of this genus may provide valuable models to obtain novel insights into host ecology and evolution [72].
As a result of two independent colonization events [8,26,53,80,91], as well as the influence of the reforming of the Dessaretes Lake system in the past [1,2,69,85,88], the Balkan Peninsula harbors remarkable species diversity among freshwater fishes belonging to the suborder Cyprinoidei.Currently, 17 highly diversified genera of cyprinoids are recognized in the Balkans.Nevertheless, some genera exhibit high morphological similarity and/or close phylogenetic relationships, i.e.Telestes and Squalius or Barbus and Luciobarbus [40,52,68].Barbus and Squalius are the genera with the highest species richness in the Balkans and are the only genera distributed across all southern European Peninsulas [53,67,68].Chondrostoma species are distributed in the northern Mediterranean drainages across Europe, western Asia, and the Middle East [21], while those of Telestes inhabit only the Balkan and Apennine Peninsulas [9,40].In contrast, the species distributions of Delminichthys, Pachychilon, Pelasgus and Tropidophoxinellus are limited to several river streams of lake systems in the Balkans [40].Although only two species of Luciobarbus (former Barbus [86,87]) are native of the Balkans, they are associated with two different evolutionary lineages.While L. albanicus (Steindachner) is phylogenetically closer to the Middle Eastern and North African species of Luciobarbus, L. graecus (Steindachner) is closely related to the Iberian lineage of Luciobarbus spp.and to L. lydianus (Boulenger) from Turkey [89].

Fish sampling
Fifty-nine individuals from seven species representing six genera of cypriniforms (Cyprinidae and Leuciscidae) were collected by means of gill nets or electro-fishing from seven localities in Bosnia and Herzegovina, Croatia, and Greece, during the period 2014-2017 (Fig. 1).Sampling was carried out under field approvals MPK BR: 07/1/625-342/15/ (Bosnia and Herzegovina), URBROJ: 517 -07 -l-l-l-16-4 (Croatia) and HCMR: 220965/2583/22-8-2011 (Greece).Fish hosts were identified in situ using the key provided by Kottelat and Freyhof [40], and the identification was subsequently confirmed using sequences of the cytochrome b mitochondrial gene recorded by Radek Šanda (National Museum, Prague, Czech Republic) and Jasna Vukić (Charles University, Prague, Czech Republic).Scientific names and the classification of fishes are those provided in Fricke et al. [25].Live fishes were kept in aerated holding tanks until they were processed for parasitological examination; fishes were sacrificed by severing the spinal cord.

Parasite sampling
Gills of freshly killed fishes were extracted, placed in a Petri dish containing water, and examined using a SZX7 stereomicroscope (Olympus, Tokyo, Japan).Monogeneans were removed from the gills using fine needles and prepared as in Řehulková [62].Specimens used for morphological examination were completely flattened under coverslip pressure in order to best expose their sclerotized structures (haptoral and reproductive sclerites), and fixed with a mixture of glycerine and ammonium picrate (= GAP; Malmberg [44]).Specimens used for DNA analysis were bisected using fine needles.Subsequently, one half of the body (either the posterior part with haptoral sclerites or anterior part containing the male copulatory organ) was fixed in 96% ethanol for later DNA extraction, and the remaining half was mounted on a slide, fixed with GAP for species identification, and kept as a hologenophore (i.e. a voucher specimen from which a molecular sample is directly derived (sensu Pleijel et al. [54]).The mounted specimens (or their parts) were studied using a BX61 microscope (Olympus, Tokyo, Japan) equipped with phase-contrast optics.Drawings of the sclerotized structures were made with the aid of a drawing attachment and edited with a graphic tablet (Wacom Intuos5 Touch) compatible with Adobe Illustrator and Adobe Photoshop (Adobe Systems Inc., San Jose, CA, USA).Measurements were taken using Stream Motion 1.9.2 image analysis software (Olympus).The list of measurements for the sclerites is shown in Figure 2. All measurements (in micrometres) are provided as the mean followed by the range and the number (n) of specimens measured in parentheses.The numbering of hook pairs (in Roman numerals I-VII) is that recommended by Mizelle [48].The male copulatory organ is henceforth abbreviated to MCO.After morphometric analysis, the specimens (or their parts) fixed with GAP were dehydrated and re-mounted in Canada balsam as permanent mounts, according to Ergens [22].Type and voucher specimens collected during the present study were deposited at the Institute of Parasitology of the Czech Academy of Sciences (IPCAS), České Budějovice, Czech Republic and the Muséum National d'Histoire Naturelle (MNHN), Paris, France as indicated in the respective species accounts.To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature [35], details of the new species have been submitted to ZooBank.Epidemiological characteristics such as parasite prevalence (percentage of infected hosts), abundance (mean number of parasites per host taking into account both infected and uninfected individuals), and intensity of infection (minimum and maximum number of parasites per infected host) were calculated for each Dactylogyrus species according to Bush et al. [10].

DNA extraction, amplification, and sequencing
Bisected Dactylogyrus individuals preserved in ethanol were dried using a vacuum centrifuge.DNA was extracted using the standard protocol (DNeasy Blood & Tissue Kit, Qiagen, Hilden, Germany).Partial 18S ribosomal DNA, the entire ITS1 region, and partial 5.8S ribosomal DNA were amplified using the primers S1 and IR8 following Šimková et al. [74].DNA amplification was performed following the protocol and conditions described in Benovics et al. [4].Partial 28S ribosomal DNA was amplified using the primers C1 and D2 following Hassouna et al. [34].DNA amplification was performed using the same conditions as described in Šimková et al. [75].The purification of PCR and subsequent sequencing follow Benovics et al. [4].Sequencing was performed on an ABI 3130 Genetic Analyzer (Applied Biosystems).Gene sequences of the new Dactylogyrus species were generated as part of larger phylogenetic studies [4,77] and have been deposited in GenBank previously (their accession numbers are in Table 1).

Phylogenetic reconstruction
Sequences of partial 18S rDNA, combined with the ITS1 region, and partial 28S rDNA were used for phylogenetic reconstruction.DNA sequences were aligned using the fast Fourier transform algorithm implemented in MAFFT [38].
Final alignments were trimmed manually to unify the length of all sequences.The final nucleotide sequence alignment contained sequences of 27 species (19 previously described, 7 new and one unnamed) of Dactylogyrus from the Balkans obtained from GenBank, and two Dactylogyrus species from Carassius gibelio (Bloch) (namely D. anchoratus (Dujardin, 1845) and  1).JModelTest 2.1.10[14,30] was employed to infer the optimal evolutionary model for each genetic segment (18S, ITS1 and 28S respectively), using the Bayesian information criterion (BIC) as the penalization algorithm.Maximum likelihood (ML) and Bayesian inference (BI) analyses were conducted in RaxML v.8.2.11 [78,79] and MrBayes 3.2.6 [66], respectively.Internal node support for the ML tree was assessed by running 1000 bootstrap pseudoreplicates.Two parallel runs, each with four Markov chains, were executed for BI analysis and run for 1,000,000 generations.Trees were sampled every 100 generations and the first 30% of the resulting trees were discarded as initial burn-in.Convergence was indicated by an average standard deviation of split frequencies per parallel run of <0.01, and subsequently checked using Tracer v. 1.7.1 [60].Posterior probabilities were calculated as the frequency of samples recovering particular clades.To uncover the genetic divergence between morphologically similar species, uncorrected pair-wise genetic distances (p-distances) were calculated for each genetic segment in MEGA7 [41].The same species as those used in phylogenetic reconstruction analyses (excluding species used as outgroup) were used for the computation of genetic distances.The final alignments contained sequences of 27 Dactylogyrus species (see Supplementary Tables 1-3).

Results
Seven cyprinoid fish species from the Balkan Peninsula were examined for monogeneans: Chondrostoma knerii  16) vitellarium coextensive with gut; (17) haptor possessing one pair of anchors (pointed to the dorsal side of the haptor) with bases connected by a dorsal bar, seven pairs of hooks with normal distribution [48], and one pair of needle-like structures lying near hooks of pair V; ventral bar present or absent.
Site on host: Gill lamellae.
Etymology: This species is named for Romulus, the legendary founder of Rome and the twin brother of Remus, and refers to the resemblance with the below-named species.
Remarks: Morphologically, Dactylogyrus romuli n. sp.belongs to the group of congeners having a ventral bar derived from the cross-shaped type, where the anterior arm is more or less bifurcated and the posterior arm (often elongated) is split or terminally frayed (= the five radial type).These include Site on host: Gill lamellae.Prevalence, mean abundance and intensity range of infection: 90% (9 fish infected/10 fish examined); 123.4; 12-687 monogeneans per infected host.
Etymology: This species is named for Remus, the legendary founder of Rome and the twin brother of Romulus, and refers to the resemblance with the above-named species.
Remarks: Dactylogyrus remi n. sp. may be confused with D. romuli n. sp. by having nearly identical haptoral structures and very similar copulatory sclerites.In the absence of comparative material, differentiation of the two species may be difficult.However, the comparative morphology of the MCO is the best means of separating the specimens and together with molecular data provide sufficient evidence that the two species are distinct.Dactylogyrus remi n. sp.can be distinguished from D. romuli n. sp. by its MCO possessing a more robust accessory piece with a cornet-shaped distal fold (the distal fold without the angularly expanded proximal portion in D. romuli n. sp.).In addition, the base of the copulatory tube appears to be more elongated in D. romuli n. sp.than in D. remi n. sp.(see Fig. 6  Site on host: Gill lamellae.
Etymology: The specific name is from Latin (recisus = short, brief), referring to the length of the copulatory tube.
Etymology: This species is named after Dr. Radek Šanda, National Museum, Prague, Czech Republic, in recognition of his extensive research on the systematics and phylogeny of Cypriniformes.
Etymology: The specific name, a noun, refers to the shape of the ventral bar resembling an octopus.
Remarks: Dactylogyrus octopus n. sp.can be assigned to the group of Dactylogyrus spp. with an inverted T-shaped ventral bar.However, the pyriform anterior arm and two transverse arms bent anterolaterally distinguish this new species from all other known members of the genus.On the basis of the morphology of the MCO, D. octopus n. sp.most resembles Dactylogyrus spp.possessing MCO of the "nanus" type [58], which is characterized by a copulatory tube with a foot-like base (usually with a heel-like apical projection) and relatively short curved shaft, and an accessory piece differentiated into two parts (the proximal part is attached to the base and the distal part is widened, guiding the terminal part of the tube).In D. octopus n. sp., the MCO differs from that of the "nanus" type  Type host and locality: Delminichthys adspersus (Heckel, 1843); Nezdravica River (Neretva Basin), Tihaljina (43°1 9 0 00.05 00 N 17°23 0 20.01 00 E), Bosnia and Herzegovina (July 2015).
Etymology: This species is named after Dr. Jasna Vukić, Charles University, Prague, Czech Republic, in recognition of his extensive research on the systematics and phylogeny of Cypriniformes.
Etymology: The specific name is from Greek (leptos = thin, slender) and refers to the shaft of the anchors.
Remarks: Dactylogyrus leptus n. sp.could be confused with Dactylogyrus vranoviensis Ergens, 1956, a gill parasite of Squalius cephalus (Linnaeus) (Danube, Oder and Elbe Rivers) [58], by having similar anchors and a similar dorsal bar.Anchors with a relatively short inner root, wide base, markedly elongate shaft with angular bend at its proximal quarter, and reduced point are common to both species.The ventral bar in the new species, although size-reduced as in D. vranoviensis,   [4,31,58].Unlike other Dactylogyrus species possessing a three-armed ventral bar, all three arms in the three species are almost the same size and are characterized by pointed ends (especially the transverse arms).The MCO of D. leptus n. sp. is intermediate between the "nanus" and "chondrostomi" types (i.e. the distal triangular widening of the accessory piece is elongated backwards along the tube to its initial part; it is short and claw-shaped [58]).It is most similar to that of Dactylogyrus folkmanovae Ergens, 1956 [58] by having an accessory piece with subterminal bifurcation but differs from it by having a more robust sheath-like terminal backwards elongation (vs.thin, hook-shaped backwards elongation in D. folkmanovae).

Genetic divergences of Dactylogyrus species
In order to clearly document the specimens from which DNA sequences were obtained in this study, morphological vouchers (hologenophores) of the newly described Dactylogyrus species were deposited in publicly accessible collections (IPCAS, MNHN).Illustrations of the MCOs directly taken from the hologenophores are presented in Figure 13.Computations of genetic distances were performed for each genetic segment individually (partial gene coding 18S rRNA, partial gene coding 28S rRNA, and the ITS1 region).Alignments of 446, 770, and 390 positions, respectively, were used for the analyses.All positions containing gaps or missing data were eliminated from the alignments.The morphologically almost indistinguishable species D. romuli n. sp. and D. remi n. sp.exhibited moderate genetic divergence (p-distance = 0.2% in 18S, 2.3% in 28S, and 5.1% in ITS1).Genetic divergence was also investigated between phylogenetically and morphologically similar Dactylogyrus spp.parasitizing Pachychilon spp.(namely D. recisus n. sp., D. martinovici and D. petkovici).Dactylogyrus recisus n. sp. was genetically distant from D. martinovici and D. petkovici by 0.2% in partial 18S, by 1.4% and 1.7% in partial 28S, respectively and by 5.6% and 7.9% in ITS1, respectively.All pair-wise genetic distances are shown in Supplementary Tables 1-3.

Phylogenetic position of the newly described Dactylogyrus species
The final concatenated alignment of DNA sequences of 29 species contained 1647 unambiguous nucleotide positions.The optimal evolutionary models were K80 + I for partial 18S rDNA (446 bp), SYM + G for the ITS1 region (410 bp) and TIM2 + I + G for partial 28S rDNA (791 bp).Both phylogenetic analyses (ML and BI -Fig.14) provided trees with congruent topologies and differed only in their node support values.Several well-to-moderately supported groups were revealed in the lineage comprising Dactylogyrus parasitizing cyprinoids.

Discussion
Recent studies on Mediterranean cypriniforms suggest that the species diversity and endemicity of these fishes is higher than previously estimated [6,7,12,16].Considering the high host specificity of Dactylogyrus spp., the discovery of new species parasitizing Mediterranean cypriniforms can likewise be expected.A recent study carried out in the Balkan Peninsula [4] revealed eight potentially new Dactylogyrus species on two species of cyprinids (L.albanicus, and L. graecus) and five species of leuciscids (C.knerii, D. adspersus, P. macedonicum, T. spartiaticus, and T. karsticus).In the present paper, seven of these species are described on the basis of integrated morphological and molecular data; the Dactylogyrus species found on S. tenellus was not identified to species level due to an insufficient number of specimens.
Two pseudocryptic Dactylogyrus species, D. romuli n. sp. and D. remi n. sp., are described from L. albanicus and L. graecus, respectively, the only two species of Luciobarbus that are native in the Balkans.The highly diversified Luciobarbus comprises more than 30 species widely distributed along rivers draining into the Persian Gulf and the Mediterranean, Caspian, and Black seas [40].To date, a total of 51 Dactylogyrus species are known from 29 species of Dactylogyrus vukicae n. sp. is described from D. adspersus.A new species was expected to be found as no monogeneans had yet been described from this host genus.Delminichthys is one of the geographically most isolated genera of leuciscids.Species of this genus inhabit small karstic streams of the central Adriatic region [24].According to Perea et al. [52], the independent evolution of Delminichthys began 14 MYA, when it separated from Pelasgus, a genus whose representatives were previously assigned to the southern Balkan Pseudophoxinus spp.[24,40].Due to a long period of separate evolutionary history [52], it is expected that species of Delminichthys host their own unique fauna of monogeneansi.e., fauna that does not occur on other genera within the Leuciscidae.Indeed, D. vukicae n. sp.possesses a morphologically unique ventral bar that cannot be assigned to any of the 16 morphological types proposed for Dactylogyrus spp.so far reported from Palaearctic fishes (see [58]).The molecular data provided in this paper shows that D. vukicae n. sp. is phylogenetically close to D. sekulovici from P. pictum.A posteriori comparison of the hologenophores of these two Dactylogyrus spp.revealed no obvious morphological resemblance between their haptoral structures.However, both species share similarity in the morphology of their MCOsspecifically, the thin-walled base of the copulatory tube is bulbous with a medial finger-like thickening.According to Perea et al. [52], D. adspersus and P. pictum are representatives of phylogenetically unrelated ancient lineages, but have a similar geographical distribution.The phylogenetic proximity of D. vukicae n. sp. and D. sekulovici suggests the host-switching of parasites between these two leuciscid hosts living in sympatry in the central Adriatic region.
Dactylogyrus leptus n. sp. is described from C. knerii, a species endemic to the Neretva River (Bosnia and Herzegovina, Croatia) [29].Species of Chondrostoma are distributed from the Rhine, Danube, and Po basins to the east, reaching southwest Iran [63].Chondrostoma knerii belongs to the Italo-Balkanic group together with the Italian Chondrostoma soetta Bonaparte and the Bosnian Chondrostoma phoxinus Heckel [63].To date, 21 Dactylogyrus species have been recorded on the gills of ten species of Chondrostoma [4,47,58].The present phylogenetic analysis showed that D. leptus n. sp.forms a well-supported group with D. rysavyi from A. thessalicus and undescribed Dactylogyrus sp. from S. tenellus, an endemic species in the Balkans.Dactylogyrus folkmanovae Ergens, 1956, parasitizing many Squalius spp. in Europe, appears in a sister position to this clade.The morphological similarities in MCOs between D. leptus n. sp. and the above-mentioned Dactylogyrus spp.from Squalius spp.(see also remarks to D. leptus n. sp.) suggest that D. leptus n. sp.originated from a host-switch from widely distributed species of Squalius, and then adapted its haptoral structures to its new host species.

Conclusion
Dactylogyrus is the most speciose genus (>900 nominal species; [28]) among helminth parasites and it is clear that the number of recorded Dactylogyrus species will continue to increase [13].Since Dactylogyrus species exhibit remarkably high host specificity, the extraordinary species richness of this super-genus seems to be predictable from the high diversity of cypriniform hosts.If we leave out the number of regions/ hosts unexplored for these parasites, a significant proportion of Dactylogyrus diversity may be hidden behind unrecognized cryptic species.The present study illustrates that some species considered as cryptic might be designated as pseudocryptic after a posteriori detailed morphological examination, as happened with D. romuli n. sp. and D. remi n. sp.In many groups, the morphological differentiation of closely related species may be difficult even for specialists, because a long time lag may exist between the primary genetic speciation and morphological differentiation [37].Thus, for accurate species characterization, it is particularly important to acquire both morphological and molecular data from the same individual specimens, ideally along with illustrations of taxonomically important structures directly taken from hologenophores.

Figure 4 .
Figure 4. Phase-contrast micrographs of the sclerotized haptoral structures of Dactylogyrus spp.Arrows indicate the shape/type of the ventral bars.(a) D. romuli n. sp.(five radial type with long terminally frayed posterior arm), (b) D. recisus n. sp.(five radial type with posterior arm shorter than anterior one and with deeply frayed termination), (c) D. sandai n. sp.(cross-shaped type with posterior arm longitudinally split into several rays), (d) D. octopus n. sp.(inverted T-shaped type with pyriform anterior arm), (e) D. vukicae n. sp.(inverted T-shaped type with anterior arm reduced into several finger-like rays), (f) D. leptus n. sp.(poorly sclerotized, inverted T-shaped type with pointed arms of the same size).

Figure 6 .
Figure 6.Phase-contrast micrographs of the MCOs in Dactylogyrus romuli n. sp.(a) and D. remi n. sp.(b).Characters of interest are indicated by arrows: (1) distal fold without expanded proximal portion, (2) medial fold on the opposite side to the distal fold, (3) base markedly elongated, (4) distal fold with expanded proximal portion (cornet shaped), (5) medial fold on the same side as the distal fold, (6) base slightly elongated.
adaptation to similar environments provided by phylogenetically closely related hosts, i.e.Tropidophoxinellus and Scardinius.

Table 1 .
List of Dactylogyrus species, their cyprinoid host species, country of collection, and GenBank accession numbers for sequences used for the phylogenetic reconstruction.
Dactylogyrus dirigerus Gussev, 1966 and Dactylogyrus elegantis Gussev, 1966, both parasites of Chondrostoma spp. in Central and Eastern Europe The first well-supported group was formed by two Dactylogyrus species from Pachychilon pictum (namely D. martinovici and D. petkovici) and D. recisus n. sp.collected from P. macedonicum.Dactylogyrus leptus n. sp.formed a well-supported group with Dactylogyrus rysavyi Ergens, 1970 from Alburnoides thessalicus Stephanidis and an undescribed Dactylogyrus sp. from Balkan S. tenellus.Dactylogyrus folkmanovae, a species parasitizing many Squalius spp. in Europe, was in basal position in this clade.Two new Dactylogyrus species from Balkan Luciobarbus spp.clustered together with D. crivellius Dupont & Lambert, 1987, a species strictly parasitizing Barbus spp.throughout Europe; however, the clade was only moderately supported using BI.All three species share similar haptoral sclerites (the same type of ventral bar).Dactylogyrus spp.(MCOs of hologenophores) parasitizing cyprinoids in the Balkan Peninsula.Dactylogyrus vukicae n. sp. was phylogenetically proximal to Dactylogyrus sekulovici Ergens, 1970 from P. pictum.Dactylogyrus sandai n. sp.grouped with D. suecicus and D. rutili (both common species of Rutilus spp.), which share similar morphological features with the newly described species.