Introduction

Species of Gyrodactylidae van Beneden & Hesse, 1832 (Platyhelminthes: Monogenoidea) from the Neotropical region are known for parasitizing characiforms, cichliforms, cyprinodontiforms, gymnotiforms, mugiliforms, perciforms and siluriforms (Boeger & Vianna 2006; Cohen et al., 2013). The siluriforms are the most diverse group of fish in South America, and are mainly found in Brazil (Reis et al., 2003). Among the Neotropical siluriforms, some species of Auchenipteridae Blekker, 1862, Callichthyidae Bonaparte, 1838, Heptapteridae Gill, 1861 and Loricariidae Rafinesque, 1815 are known to harbor only gyrodactylids (Kritsky et al., 1995; Popazoglo & Boeger, 2000; Bueno-Silva & Boeger, 2009; 2014; Razzolini et al., 2019; Vianna & Boeger 2019). Species of Gyrodactylus von Nordmann, 1832 and Scleroductus Jara & Cone, 1989, both gyrodactylids, are parasites of siluriforms in Brazil. Among the species of Gyrodactylus, eight are parasites of callichthyids. Gyrodactylus anisopharynx Popazoglo & Boeger, 2000, G. corydori Bueno-Silva & Boeger, 2009, and G. superbus (Szidat, 1970) Popazoglo & Boeger, 2000 are parasites of Corydoras paleatus (Jenyns, 1842) and C. ehrhardti Steindachner, 1910, while G. samirae Popazoglo & Boeger, 2000 exclusively parasitize C. ehrhardti (Kritsky et al., 1995; Cone et al., 2010). Gyrodactylus bueni Bueno-Silva & Boeger, 2014, G. major Bueno-Silva & Boeger, 2014, and G. scleromystaci Bueno-Silva & Boeger, 2014 have been found parasitizing Scleromystax macropterus (Regan, 1913), and S. barbatus (Quoy & Gaimard, 1824) (Bueno-Silva & Boeger, 2014). Gyrodactylus polyadenus Vianna & Boeger, 2019 is a parasite of Callichthys callichthys (Linnaeus, 1758) (Vianna & Boeger, 2019).

The Gyrodactylidae family contains 25 valid genera (Přikrylová et al., 2021), 11 of which are monotypic and exhibit the addition, absence, or modification of haptoral sclerites. These include Anacanthocotyle Kritsky & Fritts, 1970, Accessorius Jara, An & Cone, 1991, Citharodactylus Přikrylová, Shinn & Paladini, 2017, Diechodactylus Vianna, Boeger & Silva-Souza, 2008, Gyrdicotylus Vercammen-Grandjean, 1960, Gyrocerviceanseris Cone, Abbott, Gilmore & Burt, 2010, Ieredactylus Schelkle, Paladini, Shinn, King, Johnson, van Oosterhout, Mohammed & Cable, 2011, Mormyrogyrodactylus Luus-Powell, Mashego & Khalil, 2003, and Scutalatus Vianna, Boeger & Dove, 2007. Species of Diplogyrodactylus Přikrylová, Matějusová, Musilová, Gelnar & Harris, 2009 and Tresuncinidactylus Přikrylová, Barson & Shinn, 2021 present hooks with different sizes and shapes, and a male copulatory organ (MCO) consisting of a muscular pouch (Přikrylová et al., 2009; 2021). In addition to the morphological features that support the proposal of new genera, molecular data were used to confirm the status of new species for Gyrocerviceanseris, Ieredactylus, and Tresuncinidactylus (Cone et al., 2010; Schelkle et al., 2011; Přikrylová et al., 2021).

Species of five monotypic genera were proposed from Neotropical hosts: Anacanthocotyle anacanthocotyle Kritsky & Fritts, 1970 and Accessorius peruensis Jara, An & Cone, 1991 parasitizing characiforms; Diechodactylus joaberii Vianna, Boeger & Silva-Souza, 2008 and Scutalatus magniancoratus Vianna, Boeger & Dove, 2007 parasitizing gymnotiforms; and Ieredactylus rivuli Schelkle, Paladini, Shinn, King, Johnson, van Oosterhout, Mohammed & Cable, 2011 parasitizing cyprinodontiforms (Kritsky & Fritts, 1970; Jara et al., 1991; Vianna et al., 2007; 2008; Schelkle et al., 2011).

Oncoceratium n. gen. is the sixth monotypic genus from the neotropical region proposed to accommodate Oncoceratium amphidactylum n. gen. n. sp. from siluriforms, based on the exclusive features of the haptoral structures and the molecular data of rDNA.

Materials and methods

Host collection and morphological analysis

Specimens of the tamboatá fish, Hoplosternum littorale (Hancock, 1828) (Callichthyidae) were collected from February 2014 to February 2018 in the Batalha River, in the municipal district of Reginópolis, and the Peixe River and the Tietê River, in the municipal district of Anhembi, all in the state of São Paulo (coordinates in the respective description).

Fish were captured using nylon monofilament gillnets with different mesh sizes (ranging from 20 to 100 mm internodes). These were placed on slopes or near the river bottom at night, and removed before dawn (10 hours exposure). The fish were removed from the nets, anesthetized with a eugenol solution (clove oil), and submitted to euthanasia through the physical method of medullary section, before being immediately individualized in plastic bags. Part of the hosts were analyzed for parasites while still in the field, while a small number of excess hosts were frozen and taken to the laboratory on the same day, to subsequent analysis. The fish were analyzed within 24 hours after collection

Collections were carried out according to the guidelines of the scientific fishing license, under the authorization of the Chico Mendes Institute of Biodiversity (ICMBio) through the System of Authorization and Information on Biodiversity (SISBIO - authorization nº 40998-2). The research project was submitted to the Ethical Committee on Animal Use (CEUA) of the Centro Universitário Sagrado Coração (the Sagrado Coração University Center) (UNISAGRADO) (authorization nº 3353050417). According to Brazilian laws, species registration for scientific research purposes was carried out at SisGen (A7D0C01)

Specimens of gyrodactylids were collected under a dissecting microscope, preserved individually in 96% ethanol, and mounted on a slide prepared with Gray & Wess mounting medium, while others were stained with Gomori’s trichrome and mounted in Canada balsam (Humason, 1979). Drawings were prepared with the aid of a camera lucida on an Olympus BX51 microscope, equipped with phase contrast. Pictures were taken e câmera digital Olympus DP73, respectivamente acopladas em microscópio Olympus CX41.

Two specimens were processed for confocal laser scanning microscopy using a Leica TCS SP5 confocal microscope, for this, they were fixed with 70% alcohol, then stained with Gomori's trichrome and mounted between slide and cover slip and taken to the confocal microscope. The samples were imaged using laser 488 nm with fluorescence emission collected at 590 nm.

Measurements were made with ImageJ (NIH, Inc.) and are reported in micrometers (µm); the mean is followed by the range and sample size in parentheses (n). Measurements were taken by straight-line distances (Figs. 1–4).

Figs. 1–4
figure 1

Measurements of the structures of Oncoceratium amphidactylum n. gen. n. sp. (Gyrodactylidae) on the body surface infecting of Hoplosternum littorale (Callichthyidae). 1. Superficial bar: LSB - length of superficial bar; WSB - Width of superficial bar; 2. Male copulatory organ: Diameter; 3. Anchor: LA – Length of anchor; LPA – Length of point of anchor. 4. Hook: LH - length of hooklet; LSH - length of shaft of hook; LFH – Length of filament of hook loop.

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Type specimens were deposited in the helminthological collection of Museu de Zoologia da Universidade de São Paulo (the Zoological Museum of the University of São Paulo), São Paulo (MZUSP), Brazil, as described in the respective descriptions.

Molecular analysis

DNA extraction was performed with a blood and tissue kit (Qiagen, Hiden, Germany). The fragments of ITS 1, 5.8S and ITS 2 were amplified and sequenced with ITS 1 (5'-TTTCCGTAGGTGAACCT-3') and ITS 2 (5'-TCCTCCGCTTAGTGATA-3') primers, (Cunningham, 1997). The polymerase chain reaction (PCR) followed the protocol of the PureTaq Ready-to-go PCR Beads kit (GE Healthcare) with the addition of 2 µL of each primer (3.2 M), 3-4 µL of template DNA, and water to complete to a final volume 25 µL. The PCR cycle was performed with initial denaturation at 94 ºC (5 min) followed by 40 cycles as follows: denaturation at 94 °C (1 min), annealing at 50 °C (1 min), extension at 72 °C (1 min), and final extension 72 °C (5 min). The PCR results were confirmed through electrophoresis in 1.5 % agarose gel. The PCR product was purified with QIAquick PCR Purification Kit (Qiagen®, CA, USA). Automated sequencing was performed directly on the purified PCR products from specimens, using a BigDye v.3.1 Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) for cycle sequencing.

The obtained partial sequences were assembled and edited using Sequencher 4.8 software (Gene Codes Corporation) to obtain consensus sequences. The matrix was constructed with the ITS consensus sequence obtained in this study and those of 27 species, mainly from those genera with additional or modificated haptoral sclerites, available in the Genbank and with sequence obtained in this study (Table 1). All sequences were aligned using the ClustalW algorithm within MEGA X v10.0.5 (1993-2019) (Kumar et al., 2018) for distance analysis, and by ClustalW (Larkin et al., 2007) and standard settings in the Geneious 7.1.3 software (Kearse et al., 2012) for Bayesian inference (BI).

Table 1 Sequences of Gyrodactylidae used in the phylogenetic reconstruction and number of GenBank database.
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The Distance analysis was performed using the Neighbor-Joining method with 10,000 repetitions based on a Maximum Composite Likelihood (MCL) (Tamura et al., 2004) model and p-distance (Nei & Kumar, 2000) using 5.8s-ITS2 rDNA with bootstrap support. The distance analysis was performed with reduced fragment to represent interspecific distances and not the phylogenetic relationship among species. It was not possible to estimate the intraspecific distance because only one sequence of the new species was obtained.

Bayesian inference was performed using species of Gyrodactylus parasitizing callichthyid hosts as an outgroup. The nucleotide substitution model was selected from 82 possible evolutionary models, based on Akaike's information criterion, using the jModel Test 2.1 (Darriba et al., 2012), which identified the general time-reversible model with gamma distribution and invariant rates (GTR + I + G) as the model of best fit. The Bayesian inference (BI) method was used for phylogenetic analysis. MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003) was used to run BI using Markov Chain Monte Carlo (MCMC) tree searches. Two parallel runs of four simultaneous searches in the MCMC were carried out for 5 million generations each. The phylogenetic trees were used by MrBayes to estimate the subsequent probability of each node in our phylogenetic reconstruction. The phylogenetic tree obtained was visualized in Figtree 1.4.2 (Rambaut, 2014).

Results

Class Monogenoidea Bychowsky 1937


Subclass Polyonchoinea Bychowsky 1937


Order Gyrodactylidea Bychowsky 1937


Family Gyrodactylidae Van Beneden & Hesse 1863


Oncoceratium n. gen.


Etymology. The generic name originates from Greek and refers to the anchors’ shape that remembers horns (onco = hook; ceratium = diminutive of horn).


Diagnosis. Gyrodactylidae. Body divisible into cephalic region, trunk, peduncle, and haptor. Two cephalic lobes, each with head organ and spike sensillum. Cephalic glands present. Eye-spots absent. Pharynx composed of two subspherical bulbs. Two intestinal caeca not confluent. Gonads tandem; testis postgermarial. Male copulatory organ (MCO) bulbous, with one row of spinelets with similar sizes and shapes; the only spine usually found in the gyrodactylids species is absent; seminal vesicle and prostatic gland not observed. Uterus with up to two consecutive generations of embryos. Haptor with pair of ventral anchors, superficial bar rectangular, 16 hooks; hooks in two anterolateral clusters of eight hooks; the point of the anchor folded to out; deep bar absent; an only root without distinction of superficial and deep root. Parasitic of siluriforms fishes.


Oncoceratium amphidactylum n. gen. n. sp.

(Figs. 5–18)


Type host: Hoplosternum littorale (Hancock, 1828) (Callichthyidae).


Site of infestation: Body surface.


Prevalence and intensity: 22.6% and 3.1 ± 0.7


Type–locality: Batalha River (25° 50, 646′ S, 49° 43, 263′ W), Reginópolis, São Paulo, Brazil, December 2015.


Other localities: Peixe River (22° 49′ 50.7”S - 048° 06′ 37.0″ W) and Tietê River (22° 46′ 07.2″ S - 48° 08′ 27.7″ W), Anhembi municipal district, state of São Paulo.


Specimens studied: Holotype (MZUSP 8040); Paratypes (MZUSP 8041 a-k).


Etymology: The specific name originates from Greek and refers to the hooks, which are reminiscent of fingers, separated on two lobes on the haptor (amphi = on two sides; dactylum = finger).


Description: Body truncated, 558 (421–691, n = 10) long, widest at level of midlength 157 (123–227, n = 10) (Figs. 5, 10, 11). Unicellular cephalic glands, head organ conspicuous. Proximal pharyngeal bulb 83 (56–121, n = 8) in diameter, ovate; distal pharyngeal bulb 63 (51–73, n = 5) diameter, ovate. Male copulatory organ (MCO) 18 (17–19, n = 4) in diameter, bulbous, armed with one row of 5-6 spinelets (Figs. 7, 13). The single spine usually found in gyrodactylid species is absent. Seminal vesicle not observed. Gonads tandem. Testis 48.61 (n = 1) long, 35 (n = 1) wide, ovate, postgermarial and intercaecal. Germarium 29.2 (27.5–31.0, n = 2) in diameter, rounded. Uterus with up to two generations of embryos. Follicles, apparently glandular, posterior to the terminations of the caeca. Haptor 93 (63–137, n = 12) long, 134(83–196, n = 12) wide. Anchor 113 (103–125, n = 8) long; shaft slightly curved; point 24 (20–28, n = 8) long, curved outwards (Figs. 6, 9, 14, 15); superficial and deep roots undifferentiated, forming a single root (Figs. 9, 14, 15). Superficial bar 45 (37–51, n = 6) long, 14 (12–17, n = 6) wide, rectangular (Figs. 6, 14, 16, 17). Shield on the superficial bar and deep bar absent. Hooks of similar size and shape, distributed in two lobes anterolaterally disposed to the anchors, with eight hooks on each (Fig. 8, 12, 18). Hook shank 21 (17–22, n = 9) long; hooklet 7 (6–8, n = 9) long; point and shaft continuous, evenly curved; shelf concave, toe rounded; heel rounded; Filament hook loop 16 (12–23, n = 5) long (Figs. 8, 12, 18).

Figs. 5–9
figure 2

Drawings of the Holotype of Oncoceratium amphidactylum n. gen. n. sp. (Gyrodactylidae) on the body surface infecting of Hoplosternum littorale (Callichthyidae). 5. Wholemount, ventral view. 6. The complex of anchors, superficial bar. 7. MCO. 8. Hook. 9. Anchor.

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Figs. 10–14
figure 3

Photomicroscopy of Oncoceratium amphidactylum n. gen. n. sp. (Gyrodactylidae) on the body surface of Hoplosternum littorale (Callichthyidae), holotype. 10. Wholemount of adult, ventral view. 11. Wholemount ventral view of young with embryo in the uterus. 12. Hook. 13. MCO. 14. The complex of anchors, superficial bar. Objectives used in the photos: Fig. 10-11- 10X magnification; Figs. 12-13 - 100X magnification; Fig. 14 - 40X.

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Figs. 15–18
figure 4

Confocal microscopy of Oncoceratium amphidactylum n. gen. n. sp. (Gyrodactylidae) on the body surface of Hoplosternum littorale (Callichthyidae). 15. Anchor. 16. Superficial bar. 17. Haptor showing anchor, bar and hooks; 18. Details of the hooks.

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Differential morphological diagnosis

Oncoceratium amphidactylum n. gen. n. sp. is differentiated from other species of gyrodactylids by its distinctive and unique features. The horn-shaped anchor with its point curved outwards has never been observed in gyrodactylid species, including species of Gyrodactylus, which have a recurved point, forming a hook. Obviously, not all species of Gyrodactylus were compared, but in the last descriptions of species of Gyrodactylus from the Neotropical region, all species were described as having an anchor with a recurved point, forming an angle (like-hook) (Popazoglo & Boeger, 2000; Bueno-Silva & Boeger, 2009; Rubio-Godoy et al., 2010; Bueno-Silva & Boeger, 2014; García-Vásquez et al., 2015; 2018; 2019; Razzolini et al., 2019; Vianna & Boeger, 2019).

The MCO of the new species is bulbous, with one row of spinelets of similar sizes and shapes, while the unique spine usually observed in gyrodactylid species is absent or undifferentiated. Species of gyrodactylids from Neotropical region presents MCO bulbous, with spine and one or two rows of spinelets. The only species that presents an arrangement with spinelets of the same size in the only row (18-20 spinelets) is Gyrdicotylus gallieni Vercammen-Grandjean, 1960, a parasite of Xenopus laevis (Daudin) (Anura, Pipidae).

The hooks distributed in two anterolateral lobes differentiate O. amphidactylum n. gen. n. sp. from remaining gyrodactylid species, which possess hooks distributed in the anterior and posterior lobes. In the Neotropical region, species of Accessorius, Diechodactylus, Polyclithrum Rogers, 1967 and Scutalatus were described as having hooks distributed on the anterior and posterior lobes (Rogers, 1967; 1969; Vianna et al., 2007; 2008).

Representative DNA sequences and molecular diagnosis

An ITS (Internal Transcribed Spacer) rDNA sequence from O. amphidactylum n. gen. n. sp. (GenBank accession number: MT345551) was produced from one specimen.

The sequence of the new species has lengths of 691 bp, composed by partial ITS1 (261 bp), 5.8s gene (155 bp) and partial ITS2 (175 bp). The content of the sequence was A=171 (24.75%); C=128 (18.52%); G=140 (20.26%); and T=252 (36.47%); while the content of G+C = 38.78%, and the content of A+T = 61.22%. The GenBank BLAST revealed closest matches with D. martini (AM943008), followed for several species of Gyrodactylus from out of Neotropical region.

Pairwise distance analysis supports new species status for O. amphidactylum n. gen. n. sp. and indicates sufficient interspecific genetic distance to differentiate the new species from those of other genera, including Gyrodactylus. The pairwise distance based on Neighbor-Joining varied between 0.374 – 0.842 using MCL, and between 0.249 – 0.386 using p-distance. The smallest distances obtained were among O. amphidactylum n. gen. n. sp. and non-Gyrodactylus species, while the differences among species of Gyrodactylus were greatest (Table 2).

Table 2 Pairwise distance of selected species though Neighbor-joining based on the Maximum Composite Likelihood (Nei & Kumar, 2004) model (bellow of diagonal) and p-distance (above of diagonal) using ITS rDNA fragment. Bold values represent among Oncoceratium amphidactylum n. gen., n. sp. and neotropical and non-neotropical species.
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The phylograms reflect pairwise distance, where the new species clustered near Gyrodactyloides bychowskii Albova, 1948, Laminiscus gussevi (Bychowsky & Polyansky, 1953) Pálsson & Beverely-Burton, 1983 and I. rivuli (Fig. 19). This grouping is inserted in a cluster composed of species with some modification (like the shield of I. rivuli), additions (such as sclerotized plates and ribs), or the absence of haptoral structures (such as a superficial bar and shield). Species of Gyrodactylus were observed at the base of the phylogram (from neotropical callichthyids) or interspersed among Fundulotrema prolongis (Hargis, 1955) Kritsky & Thatcher, 1977, Paragyrodactylus variegatus You, King, Ye & Cone, 2014, and Swingleus ancistrus Billeter, Klink & Maugel, 2000, but distant from O. amphidactylum n. gen. n. sp. The bootstrap support value was low in MCL analysis (44) and high in the p-distance analysis (74) (Fig. 19). However, the phylograms were used to test the status of a new taxon to a new species, and not test phylogenetic position among gyrodactylids.

The Bayesian inference showed O. amphidactylum n. gen. n. sp. near all the selected species with the modification, addition, or absence of haptoral structures, and distant from the cluster composed of species of Gyrodactylus, but with low posterior probability support (0.33). Pairwise distance analysis and Bayesian inference support the new species and distance it from species of Gyrodactylus (Fig. 20).

Discussion

Siluriform fishes from the Neotropical region have been reported as hosts to several species of Gyrodactylidae, of which callichtyid fishes have the highest number of records of parasitosis by gyrodactilids. Callichtyids have been harboring only species of Gyrodactylus (Popazoglo & Boeger, 2000; Bueno-Silva & Boeger, 2009; 2014; Vianna & Boeger, 2019). In this study, Oncoceratium n. gen. is proposed to accommodate O. amphidactylum n. gen. n. sp., described from the callichthyid fish, H. littorale. Oncoceratium amphidactylum n. gen. n. sp. is differentiated from all others species of gyrodactylids by the presence of a horn-shaped anchor with its point curved outward, containing undifferentiated roots that form a continuous region.

Prior to the present study, the anchor pattern found in species of the gyrodactylid genera presented a recurved point forming an angle. Despite the difficulties of revising all Gyrodactylus species due to their high number, all species recently described in the Neotropical region have an anchor with a recurved point, forming an angle between the shaft and the point (Kritsky et al., 1995; Popazoglo & Boeger, 2000; Bueno-Silva & Boeger, 2009; 2014; Rubio-Godoy et al., 2010; García-Vásquez et al., 2015; 2018; 2019; Razzolini et al., 2019; Vianna & Boeger, 2019), as along with species of other genera. The root is one single continuous structure, without evident differences between superficial (ventral) and deep (dorsal) roots, as in species of Archigyrodactylus (Mizelle & Kritsky, 1967). Most species of gyrodactylids possess conspicuous knob-like superficial and deep roots (e.g. species of Gyrodactylus), but few species present modifications. Diechodactylus joaberii and S. magniancoratus, for example, both have a superficial root-like keel-shape structure, and an elongated deep root (Vianna et al., 2007; 2008).

Fig. 19
figure 5

Neighbor-joining phylogram of Oncoceratium amphidactylum n. gen. n. sp. (Gyrodactylidae) and of other neotropical and non-neotropical species based on the Maximum Composite Likelihood (Nei & Kumar, 2004) model (A) and based on p-distance (B) using 5.8s and partial ITS2 rDNA fragment. Bootstrap values (n = 10,000) are presented near the branches.

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The most common hook arrangement in gyrodactylids is radial at the edge of the haptor, but the hooks of O. amphidactylum n. gen. n. sp. are the same size and are distributed in two anterolateral lobes. However, in species of ten genera, different numbers of hooks are distributed in the anterior and posterior lobes of the haptor (Bakke et al., 2007), and there is the presence of further sclerites. Species of Macrogyrodactylus Malmberg, 1957 presents one pair of hooks on the anterior lobe and seven pairs on the posterior lobe (1+7). Species of Accessorius, Fundulotrema Kristky & Thatcher, 1977, Gyrocerviceanseris & Swingleus Rogers, 1969 present 3+5, while species of Gyrodactyloides Bychowsky, 1947, Lamniscus Pálsson & Beverly-Burton, 1983, Polyclithrum, and Scutalatus contain 4+4. The only species of Diechodactylus described contained 5+3 (Rogers, 1967; 1969; Kritsky & Thatcher, 1977; Pálsson & Beverley-Burton, 1983; An et al., 1991; Vianna et al., 2007; 2008; Cone et al., 2010). The African Diplogyrodactylus martini Přikrylová, Matějusová, Musilová, Gelnar & Harris, 2009 presents hooks of two different shapes and sizes, whilst the hooks of Tresuncinidactylus wilmienae Přikrylová, Barson & Shinn, 2021 possess three sizes, with the same shape (Přikrylová et al., 2009; 2021).

Oncoceratium amphidactylum n. gen. n. sp. presents an MCO bulbous, with spinelets of similar sizes and shapes distributed in a row, without the only spine usually observed in gyrodactylid species. This arrangement is very similar to that found in G. gallieni, which is a parasite of anuran (Pipidae), and presents only a row (ring) with 18-20 spinelets. Most species of gyrodactylids usually have a muscular bulbous MCO with a single spine and different numbers of rows and spinelets on each (e.g. Gyrodactylus). However, some species have been described presenting different patterns of MCO. African species are the only viviparous species known so far to have a muscular pouch-type MCO, like those of the genera Afrogyrodactylus Paperna, 1968, Citharodactylus, Diplogyrodactylus, Mormyrogyrodactylus, as well as Tresuncinidactylus, the MCO of which is an armed or unarmed muscular duct onto the muscular pouch (Přikrylová et al., 2009; Přikrylová & Luus-Powell, 2014).

Obtaining a single 5.8s and an ITS2 sequence from the new species did not allow phylogenetic position among the gyrodactylids to be established. Despite the analysis of distance not providing enough bootstrap support (Figs. 19 and 20), it showed the new species as near G. bychowskii, L. gussevi and I. rivuli, and distant from species of Gyrodactylus. The proximity of the new species to species with the addition or modification of haptoral sclerites was also observed with Bayesian inference. Gyrodactyloides bychowskii, L. gussevi and I. rivuli were observed composing a clade with Scleroductus sp. (not used in the present study) through the use of 18S rDNA (Přikrylová et al., 2021). Similar topology to the analyses made in this study was observed by Schelkle et al. (2011), which identified cluster of species with the addition of haptoral sclerites in the same clade, using only the ITS2 rDNA in Maximum likelihood analysis.

Fig. 20
figure 6

Bayesian cladogram of Oncoceratium amphidactylum n. gen. n. sp. (Gyrodactylidae) and of other neotropical and non-neotropical species based on MCMC model using 5.8s and ITS2 rDNA fragment. Posterior probability values (n = 10,000) are presented below the branches.

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The smallest pairwise distance values were among O. amphidactylum gen. n. sp. n. and non-Gyrodactylus species, suggesting greater proximity between them, but still allowing them to be differentiated from one another. However, distances were bigger among the new species and species of Gyrodactylus (Table 2), indicating that the new species does not belong to Gyrodactylus. A pairwise distance greater than 1% could indicate interspecific differentiation when associated with conspicuous differentiations, especially ecological differentiations (Ziętara & Lumme, 2003).

Oncoceratium n. gen. is monotypic, as are the vast majority of genera in Gyrodactylidae (11 of 25 genera), such as the recently proposed Tresuncinidactylus (Přikrylová et al., 2021). Afrogyrodactylus was considered monotypic for 45 years until A. girgifae Přikrylová & Luus-Powell, 2014 and A. kingi Přikrylová & Luus-Powell, 2014 were described. Three years later, A. ardae Přikrylová & Gelnar, 2017, was described. The same occurred with other species-poor genera proposed as monotypic, for which new species were identified years later, such as Paragyrodactylus Gvosdev & Martechov, 1953, Polyclithrum (proposed in 1967), Swingleus (proposed in 1969), and Scleroductus (proposed in 1989). However, Acanthoplacatus Ernst, Jones & Whittington, 2001, Fundulotrema, Gyrodactylus, and Macrogyrodactylus were proposed with the description of more than one species. Currently, more than one species is known in Afrogyrodactylus (4 spp.), Archigyrodactylus (3), Acanthoplacatus (7), Fundulotrema (6), Gyrodactyloides (4), Gyrodactylus (500+) (Pinacho-Pinacho et al., 2021), Isancistrum de Beauchamp, 1912 (2), Laminiscus Pálsson & Beverley-Burton, 1983 (2), Macrogyrodactylus (6), Paragyrodactylus (3), Polyclithrum (5), Scleroductus (3), and Swingleus (2).

The late description of species in certain monotypic genera of Gyrodactylidae may be the result of the scarcity of taxonomists with an interest in gyrodactylids, insufficient sampling, or the technological difficulty of diagnosis, and can explain the emergence of monotypic genera over several decades. From the 1950s onwards, several species were known for most genera, nevertheless, the use of molecular data in integrative diagnoses began to be used on a large scale from the 2000s, resolving some taxonomic bottlenecks in Gyrodactylidae.