Introduction
The Diplozoidae Palombi, 1949 are hermaphroditic ectoparasites fused in permanent cross-copula after maturing on a suitable host (Dos Santos and Avenant-Oldewage, Reference Dos Santos and Avenant-Oldewage2024). Diplozoids have a monoxenous life cycle and usually lay one egg at a time, from which a free-living larva (oncomiracidium) hatches and actively seeks a host. In adults, the vitellaria and the majority of the intestine are situated in the anterior part of the body, whereas the reproductive organs and the terminal segment of the gut are in the posterior. The attachment apparatus (=haptor) comprises two rows of four pairs of clamps, accompanied by a pair of small central hooks located on the ventral side of the haptor near the first pair of clamps (Khotenovsky, Reference Khotenovsky1985). Although the size and shape of the sclerotized elements in the attachment apparatus are commonly used to distinguish diplozoid species, studies have shown that clamp dimensions may vary with the size of the host fish and can also change during the ontogeny of the parasite (Matějusová et al., Reference Matějusová, Koubková, Gelnar and Cunningham2002) or between seasons (Milne and Avenant-Oldewage, Reference Milne and Avenant-Oldewage2012). Therefore, only the shape of the clamp sclerites and the length of the central hook sickle are considered reliable taxonomic characters (Bychowsky and Nagibina, Reference Bychowsky and Nagibina1959; Gläser and Gläser, Reference Gläser and Gläser1964; Khotenovsky, Reference Khotenovsky1985; Nejat et al., Reference Nejat, Benovics, Řehulková, Vukić, Šanda, Kaya, Tarkan, Abdoli, Aksu and Šimková2023).
Within Diplozoidae, two subfamilies are distinguished primarily by the number of clamp pairs on the haptor. Diplozoinae Palombi, 1949 possess four pairs of clamps and include five genera, whereas Neodiplozoinae Khotenovsky, 1980 have more than four clamp pairs on each haptor and comprise two monotypic genera (Khotenovsky, Reference Khotenovsky1985). Diplozoinae have traditionally been considered restricted to the Palearctic and Oriental regions (Khotenovsky, Reference Khotenovsky1985; Pugachev et al., Reference Pugachev, Gerasev, Gussev, Ergens and Khotenowsky2010 ), although species of Paradiplozoon Akhmerov, 1974 have recently also been reported from Africa (Avenant-Oldewage et al., Reference Avenant-Oldewage, Le Roux, Mashego and Van Vuuren2014; Dos Santos et al., Reference Dos Santos, Van Vuuren and Avenant-Oldewage2015; Benovics et al., Reference Benovics, Koubková, Civáňová, Rahmouni, Čermáková and Šimková2021). Species diversity within Diplozoidae is relatively low compared with other flatworms traditionally classified as Monogenea (currently classified as Monopisthocotyla and Polyopisthocotyla, sensu Brabec et al., Reference Brabec, Salomaki, Kolísko, Scholz and Kuchta2023). More than 70 species have been described to date (Huang et al., Reference Huang, Zhou, Yuan and Ding2023); however, only about 55 are currently considered valid (Dos Santos and Avenant-Oldewage, Reference Dos Santos and Avenant-Oldewage2020). Despite this relatively small number of recognized species, the true diversity of diplozoids is likely underestimated, particularly in poorly studied regions. Most diplozoids parasitize cyprinoid fishes, though some occur on non-cyprinoid hosts, including African Alestidae (Thomas, Reference Thomas1957; Paperna, Reference Paperna1969, Reference Paperna1979) and Cichlidae (Yildirim et al., Reference Yildirim, Zeren, Genc, Erol and Konas2010). The most speciose genus among diplozoids is Paradiplozoon, with its species exhibiting various degrees of host specificity. Some species, such as Paradiplozoon homoion (Bychowsky and Nagibina, 1959) (Benovics et al., Reference Benovics, Šimková, Papežík, Civáňová Křížová, Ondračková, Zach and Seifertová2025) and Paradiplozoon megan (Bychowsky and Nagibina, 1959) (Benovics et al., Reference Benovics, Koubková, Civáňová, Rahmouni, Čermáková and Šimková2021), have been reported from multiple host species, whereas others, including Paradiplozoon moroccoensis Koubková, Benovics and Šimková, 2021 (Benovics et al., Reference Benovics, Koubková, Civáňová, Rahmouni, Čermáková and Šimková2021) and Paradiplozoon iraqensis Al-Nasiri and Balbuena, 2016 (Al-Nasiri and Balbuena, Reference Al-Nasiri and Balbuena2016), are currently known only from a single host species and therefore appear to be more host specific. Host specificity has occasionally been used as a taxonomic criterion (Al-Nasiri and Balbuena, Reference Al-Nasiri and Balbuena2016).
Iraq is one of the richest countries in freshwater resources, including the Tigris and Euphrates rivers with their tributaries, as well as numerous lakes and marshes. Its freshwater ichthyofauna comprises 53 native species, of which 43 are cyprinoid fishes (Freyhof et al., Reference Freyhof, Kaya, Ali and Jawad2021). Currently, 24 diplozoid species, i.e., 21 Paradiplozoon spp., 2 Eudiplozoon spp. and Diplozoon paradoxum von Nordmann, 1832 (including one Paradiplozoon species as taxon inquirendum), are recorded from the country (see Table 1 and included references for Paradiplozoon spp., and Mhaisen and Abdul-Ameer (Reference Mhaisen and Abdul-Ameer2014) and Abbood and Abdul-Ameer (Reference Abbood and Abdul-Ameer2022) for records of other species). One of the generalist species, P. homoion, was recorded from 27 fish hosts, mainly belonging to Cyprinoidei. The most recent species described from Iraq are Paradiplozoon bingolensis (Civáňová et al., Reference Civáňová, Koyun and Koubková2013) from Garra rufa in Lesser Zab River (Civáňová et al., Reference Civáňová, Koyun and Koubková2013) and P. iraqensis from Cyprinion macrostomum in Tigris River (Al-Nasiri and Balbuena, Reference Al-Nasiri and Balbuena2016). The aim of the present study is to provide additional knowledge of diplozoid parasites of fishes in Al-Gharraf River, Tigris River near Thi Qar Province, where specimens of Leuciscus vorax were obtained for parasitological examination. This region represents an important environment for cyprinid fishes, in which no parasitological studies have previously been conducted.
List of previously recorded Paradiplozoon species from Iraq with the respective host records (including originally reported host species name) and references
Table 1 Long description
Paradiplozoon species recorded in Iraq are listed with their reported fish hosts and the literature sources for each host record. Several parasites are associated with many hosts and many references, especially P. pavlovskii and P. barbi, which are reported from multiple cyprinids such as Cyprinion macrostomum, Cyprinus carpio, Carasobarbus luteus, Chondrostoma regium, and Luciobarbus schejch, plus occasional non-cyprinid hosts like Silurus glanis for P. pavlovskii. Leuciscus vorax appears repeatedly as a host across multiple species, including P. ergensi, P. megan, P. minutum, P. pavlovskii, and P. rutili, and some species are specifically noted as previously recorded from that host. Other species have narrower host ranges in the listed records, such as P. megalobramae only on Cyprinus carpio, P. magnum on Paracapoeta trutta and Garra elegans, and P. zeller on Alburnus sellal. Host names include synonyms and originally reported names, so apparent differences may reflect taxonomic updates rather than distinct hosts. Reference lists vary in length by species and host, indicating uneven study effort and possible reporting bias across fish groups and locations.
Species in bold were previously recorded from Leuciscus vorax.
Materials and methods
Material collection
Fresh specimens of L. vorax (Heckel, 1843) were caught by fishermen from Al-Gharraf River during 2023, then transported in a cooling box to the laboratory and examined for ectoparasites within 24 h. The gill arches were separated in Petri dishes with saline (0.9% NaCl) and examined under a dissecting microscope (Optika S2-ST1). The polyopisthocotylans isolated and cleaned from mucosa and attached debris were fixed in hot 4% formalin for morphological study. The parasites were transported to 70% ethanol and stained with Schuberg’s Aceto carmine and mounted in Canada balsam (Palm, Reference Palm2004). For the molecular study, the cleaned parasites were fixed in 95% ethanol. The host taxonomy followed Coad (Reference Coad2010), and scientific and common names and higher-level classification were verified according to Froese and Pauly (Reference Froese and Pauly2025). Basic epidemiological data, i.e., prevalence, mean abundance and minimum and maximum intensity of infection, were calculated for diplozoid species according to Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997).
Morphological data collection
Line drawings were prepared using an Olympus BX51 (Shinjuku, Japan) light microscope equipped with phase-contrast optics and a drawing tube. Photomicrographs were taken using a digital camera attached to the microscope and processed using the Stream Motion software (version 1.9.2) (Olympus, Shinjuku, Japan). The drawings were subsequently digitized and refined using a graphic tablet (Wacom, Kazo, Japan) and Adobe Illustrator CS6 (Adobe, San Jose, USA). Measurements (in micrometres) were taken using digital image analysis in Stream Motion, and are given as the mean, with the range and number (n) of specimens measured in parentheses. The terminology for haptoral structures follows that of Owen (Reference Owen1963) and Khotenovsky (Reference Khotenovsky1985) and is used here according to the modified scheme proposed by Nejat et al. (Reference Nejat, Benovics, Řehulková, Vukić, Šanda, Kaya, Tarkan, Abdoli, Aksu and Šimková2023). 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 National Museum of Natural History (MNHN), Paris, France. To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN, 2012), details of the new Paradiplozoon species have been submitted to ZooBank.
DNA extraction, amplification and phylogenetic analyses
Diplozoid specimens selected for molecular analyses were removed from storage in ethanol and dried in a thermal block. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s standard laboratory protocol. Complete ITS2, partial 18S rDNA with ITS1, partial 28S rDNA and partial COI region were amplified using the protocols described in Benovics et al. (Reference Benovics, Šimková, Papežík, Civáňová Křížová, Ondračková, Zach and Seifertová2025). The PCR product was checked on a 1% agarose gel stained with GoodView (SBS Genentech, Beijing, China) and purified using EPPiC Fast (A&A Biotechnology, Gdansk, Poland), following the manufacturer’s protocol. Sequencing was performed bidirectionally using the PCR primers, employing commercial services provided by Macrogen Europe (the Netherlands).
Only ITS2 rDNA sequences were used for phylogenetic analyses due to their greater availability in the GenBank database. Sequences of the ITS2 region were aligned using Fast Fourier Transform in MAFFT, applying the G-INS-1 iterative refinement algorithm (Katoh et al., Reference Katoh, Misawa, Kuma and Miyata2002). To investigate the phylogenetic position of newly obtained diplozoid species, relevant available sequences of congeneric and non-congeneric diplozoids were retrieved from GenBank (see Supplementary Table S1 for a list of sequences used for phylogenetic analyses). The sequences in the final alignment were trimmed to concur with the length of diplozoid sequences obtained from GenBank. The General Time-Reversible model was applied for the entire length of the alignment. Phylogenetic reconstructions with statistical support using the maximum likelihood (ML) and Bayesian inference (BI) methods were performed in RAxML v. 8.1.21 (Stamatakis, Reference Stamatakis2014) and MrBayes v. 3.2 (Ronquist et al., Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012), respectively. The best ML tree was selected from 1000 iterations, and support for the branching pattern was validated through 1000 bootstrap pseudoreplicates. BI trees were constructed using the Metropolis-coupled Markov chain Monte Carlo algorithm. Four concurrent chains (one cold and three heated) ran for 107 generations, with trees sampled every 102 generations. The first 30% of trees were discarded as a relative burn-in period after checking that the standard deviation of split frequencies fell below 0.01. The results were checked in Tracer v. 1.7.1 (Rambaut et al., Reference Rambaut, Drummond, Xie, Baele and Suchard2018) to assess convergence. The resulting phylogram was rooted using Octomacrum europaeum Roman and Bychowsky, 1956 (as a representative of Octomacridae – phylogenetically proximal family to Diplozoidae (Sicard et al., Reference Sicard, Desmarais and Lambert2002)). To assess genetic divergence between diplozoid species, a representative ITS2 sequence was selected for each available species in GenBank. Uncorrected pairwise distances (p-distances) were then calculated using the dist.dna() function in ‘ape’ package (Paradis and Schliep, Reference Paradis and Schliep2019) in the R statistical environment, version 4.1.3 (R Core Team, 2022) and visualized in the form of a heatmap using the ‘ggplot2’ package (Wickham, Reference Wickham2016).
Results
Morphological study
Diplozoidae Palombi, 1949
Paradiplozoon Akhmerov, 1974
Paradiplozoon vorax sp. nov.
Type host: Leuciscus vorax Heckel, 1843, Cypriniformes, Leuciscidae (Leuciscinae).
Type locality: Al-Gharraf River, Tigris River, Thi Qar Province, Southern Iraq, 31°32′10.18″ N and 46°07′19.34″ E.
Other locality: Al-Gharraf River, 31°27′24.38″ N and 46°09′37.67″ E.
Site on host: Gills.
Type material: Holotype (MNHN-HE-132YT-1); six paratypes (one on the holotype slide and MNHN-HE-132YT-2–5); five paratypes (IPCAS M-849).
Infection indices: prevalence: 22% (34 infected fish/156 examined fish); mean abundance: 0.60; mean intensity of infection: 2.77 (2–5 specimens per infected host).
Representative GenBank sequence: PZ603668 (partial ITS2 region, 752 bp), PZ603670 (partial 18S rDNA and ITS1 region, 779 bp), PZ603669 (partial 28S rDNA, 863 bp), PZ603672 (COI, 419 bp).
Etymology: The specific epithet ‘vorax’ refers to the type host, Leuciscus vorax.
ZooBank registration: To comply with the regulations set out in article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN, 2012), details of the new species have been submitted to ZooBank. The Life Science Identifier (LSID) of the article is urn:lsid:zoobank.org:pub:7AB48BA9-042D-48FD-9C40-7E2507A2A910. The LSID for the new name Paradiplozoon vorax Benovics, Řehulková, Al-Kuraizi, Ali n. sp. is urn:lsid:zoobank.org:act:71C99A84-D15B-4CAC-B5F5-D3F4387F9CEA.
Description: Body X-shaped, consisting of two permanently fused adult individuals in copula (Figure 1A); total body length 3990 (3496–4630; n = 8). Fusion delineates forebody and hindbody in each individual: forebody length 2341 (1984–2986; n = 8); hindbody length 1668 (1398–1927; n = 8). Tegument with small annular transverse plicae in hindbody, numbering 30 (23–33; n = 8). Forebody fusiform, predominantly occupied by vitellarium and digestive system. Mouth subterminal, ventral, opening into buccal cavity with two bilateral buccal suckers (Figure 1B). Suckers sub-oval, muscular, with transverse septa; 62 (50–70; n = 8) long × 50 (47–55; n = 8) wide. Buccal cavity leading into pharynx, 81 (62–93; n = 8) long × 54 (46–62; n = 8) wide. Intestinal caecum with transverse diverticula along forebody, extending into hindbody without diverticula and terminating at level of fourth pair of clamps. Hindbody without lobed widening just anterior to haptor, comprising anteriorly situated reproductive organs (testis posterior to ovary) and distally located attachment organ (= haptor). Eggs ovoid, with long coiled filament attached to operculum; opercular line situated at approximately one-third of egg length measured from filament; 22 (17–26; n = 7) long × 10 (9–12; n = 7) wide (Figure 2A). Haptor armed with four pairs of clamps and one pair of central hooks located on ventral side of each haptor. Clamps arranged bilaterally in two parallel longitudinal rows (Figure 2B). All clamps wider than long; first pair of clamps slightly smaller than the other clamps. Size of clamps (n = 15): clamp I = 60 (46–93) × 75 (54–93); clamp II = 64 (46–124) × 103 (54–124); clamp III = 65 (46–132) × 109 (54–124); clamp IV = 65 (46–124) × 105 (70–124). Each clamp with sclerites arranged in a pattern characteristic of species of Paradiplozoon. Median plate U-shaped, with median groove perforated by lacunae along most of its length. Anterior half of median plate (Figure 1D-a) distally expanding into outgrowth, connected to anterior clamp jaw by two anterior joining sclerites (Figure 1D-c). Outgrowth (Figure 1D-b) deeply incised with two truncated lobes. Anterior joining sclerites elongate, L-shaped, with uniform diameter throughout their length. Posterior half of median plate (Figure 1D-j) narrowing distally before expanding into a conspicuous, collar-shaped tendon guiding termination (Figure 1D-i). Posterior half of median plate connected to posterior clamp jaw (Figure 1D-f) via two posterior joining sclerites in tandem position, both tapering distally: proximal joining sclerite (Figure 1D-h) larger than triangular distal joining sclerite (Figure 1D-g). Anterior jaw (Figure 1D-d) composed of two massive sclerites (left and right), each with a median groove extending along most of its length. Proximal longer half arched, distal minor half recurved into a wing-shaped structure. Wing-shaped part with well-developed inwardly directed spur and small joint socket for articulation with lateral sclerite (Figure 1D-e). Posterior jaw (Figure 1D-f) composed of two thin, smooth sclerites (left and right), each with thickened convex side. Two lateral sclerites (Figure 1D-e) bilaterally connecting anterior and posterior clamp jaws, each approximately half length of posterior jaw sclerite. Two central hooks (Figure 1C) located near inner margin of most posterior pair of clamps (i.e., clamp pair I); sickle length 20 (19–21; n = 12), handle length 40 (39–41; n = 12).
Paradiplozoon vorax sp. nov. ex Leuciscus vorax (Iraq). (A) Adult pair; ventral view. (B) Anterior end of body: (a) buccal sucker; (b) pharynx. (C) Central hook. (D) – Clamp III: (a) anterior half of median plate; (b) outgrowth; (c) anterior joining sclerite; (d) anterior clamp jaw; (e) lateral sclerite; (f) posterior clamp jaw; (g) distal posterior joining sclerite; (h) proximal posterior joining sclerite; (i) tendon guiding termination; (j) posterior half of median plate.
Paradiplozoon vorax sp. nov. (A) Photomicrograph of egg with filament. (B) Photomicrograph of haptor with four pairs of clamps.
Remarks
The assignment of P. vorax sp. nov. to Paradiplozoon is based on the current morphology-based classification of Diplozoinae, which includes five genera: Diplozoon Nordmann, 1832, Eudiplozoon Khotenovsky, 1985, Inustiatus Khotenovsky, 1978, Paradiplozoon Akhmerov, 1974 and Sindiplozoon Khotenovsky, 1981. Within this framework, Paradiplozoon is distinguished from the remaining genera by the absence of dilatations in the middle part of the hindbody, whereas the other genera possess hindbody dilatations of different forms (Pugachev et al., Reference Pugachev, Gerasev, Gussev, Ergens and Khotenowsky2010). The specimens examined in the present study clearly lack such dilatations and are therefore assigned to this genus. In our ITS2-based phylogenetic analyses, P. vorax sp. nov. was placed within the clade containing the type species, P. megan, although Paradiplozoon was not recovered as monophyletic (see the Molecular study section).
P. vorax sp. nov. belongs to a morphological group of Paradiplozoon species characterized by the presence of an outgrowth at the distal end of the anterior half of the median plate, connected to the anterior clamp jaw by two well-developed anterior joining sclerites. Within clade A, several species share this configuration, including P. helleni, P. homoion, P. nagibinae, P. pavlovskii and P. skrjabini, and a comparable configuration is also present in D. paradoxum (Pugachev et al., Reference Pugachev, Gerasev, Gussev, Ergens and Khotenowsky2010; Benovics et al., Reference Benovics, Koubková, Civáňová, Rahmouni, Čermáková and Šimková2021). The outgrowth of P. vorax sp. nov. is deeply incised and has truncated lobes, whereas in other species it is trapezoidal (P. skrjabini), slightly incised with rounded lobes (P. homoion, P. pavlovskii) or deeply incised with pointed lobes (P. helleni, P. nagibinae) (Pugachev et al., Reference Pugachev, Gerasev, Gussev, Ergens and Khotenowsky2010; Benovics et al., Reference Benovics, Koubková, Civáňová, Rahmouni, Čermáková and Šimková2021). The anterior joining sclerites of P. vorax sp. nov. are elongate and slender, most similar to those of P. pavlovskii and P. skrjabini, but differ from the latter two species in being L-shaped rather than straight (Pugachev et al., Reference Pugachev, Gerasev, Gussev, Ergens and Khotenowsky2010).
Of the species discussed above, P. vorax sp. nov. is morphologically most similar to P. pavlovskii, and this similarity is further supported by our ITS2-based phylogeny, in which the two species formed sister taxa (see below). Both species share (1) a relatively long hindbody, approximately two-thirds the length of the forebody, (2) the presence of multiple folds on the hindbody, (3) an intestine lacking diverticula in the hindbody and (4) clamps with anterior joining sclerites that are slender and distinctly narrower than the adjacent part of the anterior clamp jaw.
P. vorax sp. nov. differs from P. pavlovskii by having (1) a pharynx equal to or slightly larger than the suckers (vs. smaller than those in P. pavlovskii), (2) a lower number of folds in the hindbody (23–33 vs. more than 40 in P. pavlovskii), (3) a smooth postovarian part of the intestinal caecum (vs. postovarian part with irregular margins in P. pavlovskii), (4) L-shaped anterior joining sclerites (vs. straight anterior joining sclerites in P. pavlovskii) and (5) overall smaller clamps, with the upper limit of their size range overlapping the lower limit of the size range in P. pavlovskii (Pugachev et al., Reference Pugachev, Gerasev, Gussev, Ergens and Khotenowsky2010).
Molecular study
Phylogenetic tree of 78 sequences of 27 diplozoid species reconstructed by Bayesian inference. The tree is based on 644 bp long sequences from the ITS2 region and rooted using Octomacrum europaeum as outgroup. Circles at the nodes indicate posterior probabilities (upper half) and bootstrap support values (lower half) according to the legend in the upper corner. Numbers in brackets indicate the number of sequences in the collapsed branch forming the monophyletic group. The letters in boxes indicate specific lineages which are referred to in the result section.
Figure 3 Long description
The phylogenetic tree displays evolutionary relationships among 27 diplozoid species, reconstructed using Bayesian inference. The tree is rooted with Octomacrum europaeum as the outgroup. Branches are labeled with species names and numbers in brackets indicate the number of sequences in collapsed branches forming monophyletic groups. The tree is divided into six clades labeled A to F. Paradiplozoon vorax sp. nov. is highlighted in red within clade A. Circles at the nodes represent posterior probabilities and bootstrap support values, with black indicating 1.00 posterior probability and 100 percent bootstrap support, gray for 0.99 to 0.90 posterior probability and 99 to 75 percent bootstrap support and white for 0.89 to 0.80 posterior probability and 74 to 50 percent bootstrap support. The scale bar represents genetic distance of 0.08.
The final ITS2 alignment for assessing the phylogenetic position of newly described species among investigated diplozoids, was built from 79 sequences and spanned 644 unambiguously aligned nucleotide positions. The ML and BI analyses generated trees with congruent topologies and therefore only the BI tree is presented in Figure 3. The diplozoid species were divided into six well-supported clades. Paradiplozoon vorax sp. nov. was positioned within clade A with the majority of other congeners and D. paradoxum. The phylogenetic analyses revealed a well-to-moderately supported (posterior probability = 0.92, bootstrap support = 68) sister position of P. vorax sp. nov. to P. pavlovskii collected from asp, Leuciscus aspius (Linnaeus, 1758) in the Morava River (Czechia), although moderate genetic differentiation between these two species was observed (12%). A final alignment to assess interspecific genetic distances spanned 641 base pairs and included 28 diplozoid species. After the complete deletion of the positions containing gaps, a total of 463 positions were included in the final dataset. The p-distances ranged from 1% to 39%, with the highest divergence being between Inustiatus inustiatus (Nagibina, 1965) and P. opsariichthydis (Jiang, Wu and Wang, 1984) (all values are presented in the Supplementary Figure 1). In contrast to the relationship observed within the phylogenetic tree, after the deletion of sites containing gaps, P. vorax sp. nov. was genetically most similar to P. skrjabini Akhmerov, 1974 (11% dissimilarity in ITS2).
Discussion
Morphological characters derived from the sclerotized structures of diplozoids, especially the sclerites of clamp III and central hooks, are considered the most reliable for species delimitation in Diplozoidae (e.g. Price, Reference Price1967; Khotenovsky, Reference Khotenovsky1985; Matějusová et al., Reference Matějusová, Koubková and Cunningham2004; Pugachev et al., Reference Pugachev, Gerasev, Gussev, Ergens and Khotenowsky2010). However, several studies have demonstrated substantial morphometric variability in these structures, which may complicate such delimitation (e.g. Le Brun et al., Reference Le Brun, Renaud and Lambert1988). Consequently, molecular data, most notably ITS2 sequences, have become increasingly important in diplozoid taxonomy (e.g. Sicard et al., Reference Sicard, Desmarais, Vigneux, Shimazu, Lambert, Combes and Jourdane2003; Matějusová et al., Reference Matějusová, Koubková and Cunningham2004; Civáňová et al., Reference Civáňová, Koyun and Koubková2013; Benovics et al., Reference Benovics, Koubková, Civáňová, Rahmouni, Čermáková and Šimková2021; Nejat et al., Reference Nejat, Benovics, Řehulková, Vukić, Šanda, Kaya, Tarkan, Abdoli, Aksu and Šimková2023).
In the present study, P. vorax sp. nov. was recovered as the sister species of P. pavlovskii in the ITS2 phylogeny, while consistently differing in several morphological characters, most clearly in the morphology of the clamp sclerites. In addition, soft-body characters, including hindbody proportions and the number of hindbody folds, further supported the distinctiveness of the new species. At the same time, P. vorax sp. nov. and P. pavlovskii were the only species within clade A in which the intestinal caeca lacked diverticula in the postovarian part of the body, a feature that may be consistent with their close phylogenetic relationship.
Nevertheless, comparative evaluation of morphological similarity among Paradiplozoon species is often hindered by incomplete original descriptions. In many species, detailed information on soft-body morphology, which should form part of species descriptions, is absent or only briefly documented, and species descriptions are frequently based primarily on clamp sclerites, particularly those of clamp III (e.g. Civáňová et al., Reference Civáňová, Koyun and Koubková2013; Benovics et al., Reference Benovics, Koubková, Civáňová, Rahmouni, Čermáková and Šimková2021; Nejat et al., Reference Nejat, Benovics, Řehulková, Vukić, Šanda, Kaya, Tarkan, Abdoli, Aksu and Šimková2023). Consequently, evaluation of whether morphological similarities among diplozoid species reflect shared ancestry or convergent evolution remains difficult when comparisons rely predominantly on clamp characters alone.
The present ITS2 phylogeny is also consistent with previous studies showing that Paradiplozoon, as currently defined on morphological grounds, does not form a monophyletic group (e.g. Matějusová et al., Reference Matějusová, Koubková and Cunningham2004; Benovics et al., Reference Benovics, Koubková, Civáňová, Rahmouni, Čermáková and Šimková2021; Nejat et al., Reference Nejat, Benovics, Řehulková, Vukić, Šanda, Kaya, Tarkan, Abdoli, Aksu and Šimková2023). This incongruence between morphology-based classification and molecular phylogeny indicates that the generic boundaries within Diplozoidae will likely require systematic revision based on integrative datasets combining morphology and molecular evidence. Although ITS2 has become the most widely used molecular marker in diplozoid taxonomy and has substantially improved species delimitation compared with morphology alone, reliance on a single ribosomal marker remains insufficient for resolving deeper phylogenetic relationships and rigorously testing species boundaries. Ribosomal loci may provide limited resolution among closely related taxa or, conversely, overestimate divergence because of concerted evolution and lineage-specific rate variation. As demonstrated by Benovics et al. (Reference Benovics, Šimková, Papežík, Civáňová Křížová, Ondračková, Zach and Seifertová2025) and Truter et al. (Reference Truter, Přikrylová and Smit2026), commonly used ribosomal and mitochondrial markers in polyopisthocotylans may evolve at different rates and can produce phylogenetic trees differing in branch lengths, nodal support and even overall topology. Consequently, future studies of Diplozoidae should increasingly incorporate multilocus datasets combining nuclear and mitochondrial markers to obtain more robust phylogenetic hypotheses and to better evaluate patterns of host-associated diversification. Such integrative multilocus approaches will likely be necessary for a comprehensive systematic revision of Paradiplozoon and related diplozoid genera.
At the species level, the inferred phylogenetic relationship between P. vorax sp. nov. and P. pavlovskii is currently based on comparison with the only available ITS2 reference sequence of P. pavlovskii, obtained from a specimen collected from L. aspius in the Morava River, Czech Republic (AJ300714). As L. aspius is distributed widely across Europe and western Asia, whereas L. vorax is centred in the Orontes-Tigris-Euphrates region (Fricke et al., Reference Fricke, Eschemeyer and Van der Laan2026), the phylogenetic proximity of P. vorax sp. nov. and P. pavlovskii may indicate parapatric divergence within a broader leuciscid-associated parasite lineage, with P. pavlovskii historically dispersing westward with ancestral Aspius-like hosts, while P. vorax sp. nov. persisted in the Middle East. However, this interpretation remains speculative as no molecular data are currently available for Iraqi specimens identified as P. pavlovskii. Consequently, the 12% ITS2 divergence observed between these two species may reflect differentiation from the Czech population only, and Middle Eastern populations assigned to P. pavlovskii may prove substantially closer to P. vorax sp. nov., or even represent an unrecognized species complex.
Notably, there is an unusually rich diversity of Paradiplozoon species harboured by L. vorax. The newly described P. vorax sp. nov. represents the tenth species of Paradiplozoon recorded from this host and the 21st reported from Iraq overall (see Table 1 for previously recorded Paradiplozoon species in Iraq and particular species reported for L. vorax). Only one species, Paradiplozoon kasimii (Rahemo, Reference Rahemo1980), originally described as Diplozoon kasimii, is currently considered a species inquirenda because its original description lacks sufficient morphological detail for reliable comparison with congeners (Khotenovsky, Reference Khotenovsky1985; Pugachev et al., Reference Pugachev, Gerasev, Gussev, Ergens and Khotenowsky2010). Such richness is remarkable among diplozoids, as they are generally considered to be relatively host-specific (Khotenovsky, Reference Khotenovsky1985; Dos Santos and Avenant-Oldewage, Reference Dos Santos and Avenant-Oldewage2020; Nejat et al., Reference Nejat, Benovics, Řehulková, Vukić, Šanda, Kaya, Tarkan, Abdoli, Aksu and Šimková2023). It is possible that with its broad geographic range, L. vorax may increase its exposure to regionally differentiated parasite faunas, which would be in line with Gregory’s (Reference Gregory1990) hypothesis that hosts with wider distributions harbour richer parasite communities due to broader contact zones with other phylogenetically close hosts. L. vorax is currently recognized as a valid species distributed across the Tigris and Euphrates basins and, according to current catalogues, also in parts of the Orontes basin, with records from Turkey, Syria, Iraq and Iran. Older Iraqi records under Aspius vorax refer to the same currently accepted taxon, L. vorax. Thus, the historical host records summarized from Iraq do not appear to reflect host-name instability alone, but rather repeated findings on the same fish species across a broad hydrographic area. A host with such a wide distribution can encounter different local pools of polyopisthocotylan parasites, which may promote the accumulation of multiple congeners across its range rather than within a single panmictic host population. Furthermore, it is clear that not all records from L. vorax represent strict host specialists. Some diplozoids recorded in Iraq, especially P. homoion, are well-known generalists or at least broad host users within Cyprinoidei (Gelnar et al., Reference Gelnar, Koubková, Pláňková and Jurajda1994; Matějusová et al., Reference Matějusová, Koubková, Gelnar and Cunningham2002; Benovics et al., Reference Benovics, Šimková, Papežík, Civáňová Křížová, Ondračková, Zach and Seifertová2025). Similarly, some Iraqi records of P. pavlovskii suggest a broad host spectrum in the country. Therefore, a portion of the Paradiplozoon diversity reported from L. vorax may have accumulated through host sharing with sympatric cyprinoids rather than strict long-term codiversification. Related to this, it remains unclear whether multiple Paradiplozoon species are commonly present within a single local population of L. vorax. However, at least one regional survey from the Euphrates in Al-Anbar reported several Paradiplozoon taxa from L. vorax in the same study, suggesting that local co-occurrence of multiple species is indeed possible, even if the exact degree of syntopy was not demonstrated specimen-by-specimen (Al-Salmany and Al-Nasiri, Reference Al-Salmany and Al-Nasiri2022). Even so, the occurrence of so many diplozoids on a single host species remains unusual and deserves targeted sampling of multiple L. vorax populations across Iraq, ideally with combined host and parasite population genetics.
It is possible that the currently recognized diversity of Iraqi diplozoids is inflated. This possibility is strengthened by the fact that many historical Iraqi records were based on limited morphological documentation, often lacking detailed illustrations of diagnostic structures. Because clamp dimensions may vary during ontogenesis, between seasons and with host size, morphology-only identifications can be problematic in diplozoids (Matějusová et al., Reference Matějusová, Koubková, Gelnar and Cunningham2002; Milne and Avenant-Oldewage, Reference Milne and Avenant-Oldewage2012; Nejat et al., Reference Nejat, Benovics, Řehulková, Vukić, Šanda, Kaya, Tarkan, Abdoli, Aksu and Šimková2023). Therefore, some older records from L. vorax, especially those lacking voucher material or DNA data, should be interpreted cautiously until re-examined using integrative taxonomy. Nevertheless, the high number of diplozoid species reported from Iraq in general is consistent with the exceptional evolutionary history of Middle Eastern freshwaters. Iraq harbours a rich assemblage of endemic and phylogenetically distinctive freshwater fishes, with cyprinoids being one of the dominant components of the fauna (Freyhof et al., Reference Freyhof, Kaya, Ali and Jawad2021). Recent integrative surveys of Iraqi polyopisthocotylans and monopisthocotylans have likewise shown that local parasite faunas are far more diverse than previously assumed and include numerous endemic or previously undescribed taxa (Benovics et al., Reference Benovics, Rahmouni, Řehulková, Nejat and Šimková2024; Nejat et al., Reference Nejat, Benovics, Šanda, Vukić, Kaya, Tarkan and Šimková2025). This pattern fits the broader view of the Middle East as a biogeographical crossroads between Europe, Asia and Africa, where repeated dispersal and isolation events have promoted diversification of fishes and their host-associated parasite lineages.
Conclusion
The unusually high number of Paradiplozoon species reported from L. vorax identifies this host as a potentially informative model for future studies of host specificity, parasite diversification and the historical biogeography of diplozoid parasites and their hosts in Middle Eastern freshwater systems. At the same time, the present study demonstrates persistent limitations in diplozoid taxonomy, where species descriptions are frequently based predominantly on clamp sclerites, particularly those of clamp III, whereas soft-body morphology and other potentially informative characters remain insufficiently documented. More comprehensive morphological descriptions together with molecular data from multiple genetic markers will be necessary to improve species delimitation, clarify phylogenetic relationships and strengthen the systematic framework of Diplozoidae.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0031182026102479.
Data availability statement
The type specimens and vouchers are available at the Institute of Parasitology of the Czech Academy of Sciences, České Budějovice, Czech Republic, under accession number IPCAS M-849, and the National Museum of Natural History (MNHN), Paris, France, under accession number MNHN-HE-132YT-1–5. The novel sequence data of Paradiplozoon vorax sp. nov. were deposited in GenBank and are available under accession numbers PZ603668–PZ603670 and PZ603672.
Acknowledgements
We are thankful to Dr Basem A. A. Al-Sahlany (Directorate of Thi Qar, Ministry of Agriculture) for assistance with the field fish collection and Dr Ali A. Al-Darwesh (College of Veterinary Medicine, University of Kufa) for advice on laboratory techniques. We are also indebted to Prof Dr Furhan T. Mhaisen (Sweden) for allowing us to use the records of Paradiplozoon spp. in Iraq from his unpublished book.
Author contributions
A.H.A. and M.B. conceived and designed the study. M.H.K.Al.-K. and A.H.A. examined fish and collected parasitological material. E.R. performed morphological analyses and drew and described a new species. M.B. performed laboratory procedures and phylogenetic analyses. M.B., E.R. and A.H.A. wrote the draft of the manuscript. All authors substantially contributed to the final draft and approved the final version of the manuscript.
Financial support
The molecular part of this study was financially supported by the Slovak Research and Development Agency under contract APVV-23-0015.
Competing interests
The authors declare no conflicts of interest.
Ethical standards
All applicable institutional, national and international guidelines for the care and use of animals were followed.
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