Introduction
The family Gyrodactylidae Cobbold, 1864 contains 36 genera and approximately 700 species of hyperviviparous species (WoRMS 2025). In this study, we concentrate on the genus Scleroductus Jara & Cone, Reference Jara and Cone1989 (Monogenea: Gyrodactylidae), which includes gyrodactylids characterized by bulbous male copulatory organs armed with sclerotized ribs within the ejaculatory duct, infecting Neotropical catfishes (Jara and Cone Reference Jara and Cone1989; Kritsky et al. Reference Kritsky, Boeger, Mendoza-Franco and Vianna2013). To date, three species of Scleroductus have been described: S. yuncensi Jara & Cone, Reference Jara and Cone1989, the type species collected in Peru from pimelodid catfish, Pimelodella yuncensis Steindachner, 1902 (Pimelodidae) (Jara & Cone, Reference Jara and Cone1989); S. angularis Kritsky, Boeger, Mendoza-Franco & Vianna, Reference Kritsky, Boeger, Mendoza-Franco and Vianna2013, infecting barred sorubim, Pseudoplatystoma fasciatum (Linnaeus, 1766) (Pimelodidae) in Brazil (Kritsky et al. Reference Kritsky, Boeger, Mendoza-Franco and Vianna2013); and S. lyrocleithrum Kritsky, Boeger, Mendoza-Franco & Vianna, Reference Kritsky, Boeger, Mendoza-Franco and Vianna2013, from pale catfish (also known as Guatemalan chulín), Rhamdia guatemalensis (Günther, 1864) (Heptapteridae), in the Yucatán peninsula, Mexico (Kritsky et al. Reference Kritsky, Boeger, Mendoza-Franco and Vianna2013). Although members of Scleroductus have been reported infecting different catfishes throughout South America (e.g., S. yuncensi infecting white catfish, Pimelodus albicans (Valenciennes, 1840) in the provinces of Santa Fe and Buenos Aires, Argentina (Chemes et al. Reference Chemes, Takemoto and Sottini2008; Gutiérrez and Martorelli Reference Gutiérrez and Martorelli1999), and Bloch’s catfish, Pimelodus blochii Valenciennes, 1840, in Acre state, Brazil (Negreiros et al. Reference Negreiros, Tavares-Dias and Pereira2019)), the majority are referred to generically as Scleroductus sp. ((e.g., infecting South American catfish, Rhamdia quelen (Quoy & Gaimard, 1824) and Pimelodus pohli Ribeiro & Lucena, 2006 in Paraná state, Brazil (Ferrari-Hoeinghaus et al. Reference Ferrari-Hoeinghaus, Takemoto, Oliveira, Makrakis and Baumgartner2006; Sabas and Brasil-Sato Reference Sabas and Brasil-Sato2014), Pimelodus maculatus Lacépède, 1803 in the states of Rio de Janeiro and São Paulo, Brazil (dos Santos et al. Reference dos Santos, de CastroLemos-Pita and Brasil-Sato2007; Negrelli et al. Reference Negrelli, Iannacone, Abdallah and De Azevedo2021), or atipa, Hoplosternum littorale (Hancock, 1828) and trahira, Hoplias malabaricus (Bloch, 1794) in São Paulo state, Brazil (Dias et al. Reference Dias, Alves, Da Silva, Abdallah and De Azevedo2017; Gião et al. Reference Gião, Pelegrini, De Azevedo and Abdallah2020)).
In GenBank, only one sequence of 18S rDNA of Scleroductus sp. collected from R. quelen in Brazil is available (Kritsky et al. Reference Kritsky, Boeger, Mendoza-Franco and Vianna2013). Despite the scarcity of information, the genus Scleroductus has been shown to be phylogenetically closely associated to other viviparous members of the family Gyrodactylidae (Boeger et al. Reference Boeger, Kritsky, Patella and Bueno-Silva2021). Previous phylogenetic analyses of Scleroductus using 18S rDNA (Boeger et al. Reference Boeger, Kritsky, Patella and Bueno-Silva2021; Ciccheto et al. Reference Ciccheto, Razzolini, de Buron and Boeger2023; Kritsky et al. Reference Kritsky, Boeger, Mendoza-Franco and Vianna2013) suggested that Scleroductus; Ieredactylus Schelkle, Paladini, Shinn, King, Johnson, van Oosterhout, Mohammed & Cable, 2011; Gyrodactyloides Bychowsky, 1947; and Laminiscus Pálsson & Beverley-Burton, 1983 are closely related.
Here, we describe two new species of Scleroductus collected from Neotropical catfishes (Siluriformes) in Mexico; provide systematized morphometric data of the parasites’ attachment organ (haptor), by adapting the point-to-point measurements for the haptoral sclerites proposed by Shinn et al. (Reference Shinn, Hansen, Olstad, Bachmann and Bakke2004) to characterize members of the genus Gyrodactylus von Nordmann, 1832; and assess phylogenetic relationships of the new taxa within the Gyrodactylidae employing 18S rDNA sequences.
Materials and methods
Sample collection and preparation
Between 2013 and 2018, we collected two species of heptaterids in southern Mexico: pale catfish, Rhamdia guatemalensis in Río La Antigua, Apazapan, Veracruz (n = 9); and filespine chulín, Rhamdia laticauda (Kner, 1858) in Río Chiquito, Cuicatlán, Oaxaca (n = 5). Fish were collected by electrofishing and anaesthetized by an overdose of 2-phenoxyethanol (Sigma-Aldrich, Missouri, USA), prior to their preservation in bottles containing 96% ethanol. In the lab, preserved fish were inspected under a dissection microscope (Leica EZ4; Leica, Wetzlar, Germany), and worms were separated from their fish hosts using surgical needles. Worms dislodged from fish during transportation were recovered from the bottom of the bottles. Individual worms were processed for morphological and molecular analyses. Two different groups of worms were prepared to visualize their morphology: to examine internal structures (male copulatory organ (MCO) and pharynx), whole worms were cleared and mounted on a microscope slide with a drop of Grey & Wess solution, coverslipped, and incubated overnight at 60°C (Vidal-Martínez et al. Reference Vidal-Martínez, Aguirre-Macedo, Scholz, González-Solís and Mendoza-Franco2001); and to study hook morphology (hamuli, marginal hooks, ventral and dorsal bars), parasite haptors were excised and partially digested with Proteinase-K (Harris and Cable, Reference Harris and Cable2000) to remove tissue enclosing the haptoral armature (García-Vásquez et al. Reference García-Vásquez, Pinacho-Pinacho, Guzmán-Valdivieso, Calixto-Rojas and Rubio-Godoy2023). Digestion was arrested by adding a 50:50 formaldehyde:glycerine solution, and samples were coverslipped and sealed with nail varnish. For further molecular analyses, parasite bodies whose haptors had been dissected were preserved in 96% ethanol and stored individually in 1.5-mL Eppendorf tubes kept at -20°C.
Morphometric analysis
Mounted specimens were studied with a Leica DM750 compound microscope using phase contrast to analyse the partially digested haptoral hard parts. For the haptoral hard parts, pictures were taken using imaging analysis software LAS V4.12 in a Leica ICC50 HD camera, attached to a Leica DM 750 compound microscope. Using the ImageJ 1.46r software, attachment hook and body (pharynx, MCO, and haptor) measurements were taken on images. A total of 31 point-to-point measurements were taken on each specimen by adapting measurements proposed for members of the genus Gyrodactylus by Shinn et al. (Reference Shinn, Hansen, Olstad, Bachmann and Bakke2004), which include measurements of Scleroductus detailed by Kritsky et al. (1995) (see Table 1 and Figure 1 for hamulus measurements, Figure 2 for dorsal bar and ventral bar measurements, and Figure 3 for marginal hook measurements). All measurements are given in micrometres, showing average ± standard deviation, minima and maxima in parentheses. Specimens of Scleroductus species described previously from Peru (S. yuncensi) and Brazil (S. angularis and the undescribed specimen recorded by Kritsky et al. (Reference Kritsky, Boeger, Mendoza-Franco and Vianna2013)) could not be compared directly, as we could not get access to type material; instead, we contrasted the new material collected to the original descriptions from published studies. The only type material available for inspection was S. lyrocleithrum (accession no. CNHE7619–27) from Colección Nacional de Helmintos, Universidad Nacional Autónoma de México, Mexico.
Morphological measurements of Scleroductus sp. collected from Neotropical catfishes (Siluriformes) in Mexico. Scleroductus veracruzano n. sp. collected from pale catfish, Rhamdia guatemalensis in Río La Antigua, Apazapan, Veracruz; and Scleroductus oaxaqueno n. sp. collected from filespine chulín, Rhamdia laticauda in Río Chiquito, Cuicatlán, Oaxaca. Measurements of Scleroductus lyrocleithrum collected from R. guatemalensis in Yucatán, Mexico, taken from Kristky et al. 2013.
1 HTL Hamulus total length; HA Hamulus aperture; APSW Hamulus proximal shaft width; APL Hamulus point length; ADSW Hamulus distal shaft width; ASL Hamulus shaft length; AICL Hamulus inner curve length; AAA° Hamulus aperture angle; APCA° Hamulus point curve angle; IAAA° Inner Hamulus aperture angle; ARL Hamulus root length; VBL Ventral bar length; VBW Ventral bar width; VBPML Ventral bar process to mid length; VBML Ventral bar median length; VBPL Ventral bar process length; VBMemL Ventral bar membrane length, DBL Dorsal bar length; DBW Dorsal bar width; DBAPTL Dorsal bar attachment point length; MHTL Marginal hook total length; MHSL Marginal hook shaft length; MHSiL Marginal hook sickle length; MHSiPW Marginal hook sickle proximal width; MHToeL Marginal hook toe length; MHSiDW Marginal hook sickle distal width; MHA Marginal hook aperture; MHFL Marginal hook filament loop.
Light micrographs of the hamuli of Scleroductus specimens described in the present study, illustrating the point-to-point morphometric parameters measured. Measurements were adapted from those proposed for members of the genus Gyrodactylus by Shinn et al. (Reference Shinn, Hansen, Olstad, Bachmann and Bakke2004), including those from Scleroductus detailed by Kritsky et al. (1995).
Light micrographs of the dorsal bar and the ventral bar of Scleroductus specimens described in the present study, illustrating the point-to-point morphometric parameters measured. Measurements were adapted from those proposed for members of the genus Gyrodactylus by Shinn et al. (Reference Shinn, Hansen, Olstad, Bachmann and Bakke2004), including those from Scleroductus detailed by Kritsky et al. (1995).
Light micrographs of the marginal hooks of Scleroductus specimens described in the present study, illustrating the point-to-point morphometric parameters measured. Measurements were adapted from those proposed for members of the genus Gyrodactylus by Shinn et al. (Reference Shinn, Hansen, Olstad, Bachmann and Bakke2004), including those from Scleroductus detailed by Kritsky et al. (1995).
Molecular analysis
Total genomic DNA was isolated from eight specimens of Scleroductus. For genomic DNA extraction, we used DNeasy®Blood & Tissue Kit (Qiagen, California, USA) and DNAzol reagent (Molecular Research Center, Ohio, USA) following the manufacturer’s instructions. The ITS rDNA region was amplified using the primer pairs: BD1 (5′-GTCGTAACAAGGTTTCCGTA-3′) and BD2 (5′-ATCTAGACCGGACTAGGCTGTG-3′) (Bowles et al. Reference Bowles, Blair and McManus1995; Bowles and McManus Reference Bowles and McManus1993). For amplification of the 18S rDNA fragment, we used the primers SSUF (5′-GATCCTTCTGCAGGTTCACCTAC-3′) and SSUR (5′- AAGCTGGTTGATCCTGCCAGT-3′) ((Přikrylová et al. Reference Přikrylová, Vanhove, Janssens, Billeter and Huyse2013). Polymerase chain reaction (PCR) amplifications were carried out in 25-μl reactions, containing 1 μl of each primer (10 μM), 2.5 μl of 10X buffer (200 mM Tris-HCL (pH8.4), 500 mM KCL), 1.5 μl of MgCl2 (50 mM), 0.5 μl of dNTP mixture (10 mM), 0.125 μl of Platinum Taq DNA polymerase (1 U/μl) (Invitrogen Corporation, São Paulo, Brazil), and 2 μl of diluted template DNA. The following amplification profiles were used: for ITS, denaturation at 94°C for 1 min, 35 cycles of 94°C for 1 min; annealing at 50°C for 1 min; extension at 72°C for 1/ min; and a final extension of 75°C for 10 min. For 18S rDNA: denaturation at 95°C for 3 min, 30 cycles of 94°C for 1 min; annealing at 50°C for 1 min; extension at 72°C for 1.5 min; and a final extension of 75°C for 10 min. Amplicons were visualized on GelRed (Biotium, California, USA) stained 1% agarose gel. The samples were purified and sequenced in Macrogen (https://dna.macrogen.com/). Contigs were assembled and base calling differences were resolved using Geneious 8.1.8. Sequences generated were deposited in GenBank.
Alignment and phylogenetic analysis
All the sequences of ITS rDNA and 18S rDNA obtained for this study were compared in BLAST, and only sequences of 18S rDNA of Gyrodactylidae and Oogyrodactylidae available in the GenBank database (Supplementary Table S1) were included in the alignment. Species of Udonella spp. were used as out-group as proposed in Boeger et al. (2020). The 18S rDNA data set was aligned using the software ClustalW implemented in MEGA version 7.0 (Kumar et al. Reference Kumar, Stecher and Tamura2016). The best-fitting model of nucleotide substitution and parameter settings were obtained using jModeltest 2.1.10 (Darriba et al. Reference Darriba, Taboada, Doallo and Posada2012). Phylogenetic trees were inferred using Maximum Likelihood (ML) and Bayesian Inference (BI) analyses. For the first analyses, we used RAxML v7.0.4 (Stamatakis Reference Stamatakis2006) to obtain a tree using the GTRCAT substitution model with 1,000 bootstrap replicates under the rapid bootstrap algorithm. The second analysis was inferred with MrBayes v3.2 (Ronquist et al. Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012), where Metropolis-coupled Markov chain Monte Carlo (MC3) simulations were run for 10 million generations and sampled every 1,000 generations, and the first 2,500 samples were discarded as burn-in (25%). The intra- and interspecific variation of ITS and 18S among species of Scleroductus were estimated using uncorrected p-distances with MEGA 7.0.4 (Kumar et al. Reference Kumar, Stecher and Tamura2016).
Collection permits
To conduct this work, permits were obtained from the Mexican Ministries of Agriculture, Livestock, Rural Development, Fisheries and Food (SAGARPA) and of the Environment and Natural Resources (SEMARNAT) under scientific collection permits DGOPA/01173/120208.0107 and SGPA/DGVS/02967/14, respectively.
Nomenclatural acts: This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the International Code of Zoological Nomenclature. The ZooBank Life Science Identifiers (LSIDs) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix ‘http:/zoobank.org/’. The LSID for this publication is: urn:lsid:zoobank.org:pub:C61ACE0D-7528-421B-89CD-5476206BEFAF.
Results
Class Monogenea van Beneden, 1858
Order Gyrodactylidea Bychowsky, 1937
Family Gyrodactylidae Cobbold, 1864
Description of Scleroductus veracruzano n. sp. Figures 4 and 5; Table 1
Light micrographs of Scleroductus species in Mexico. (a) Scleroductus veracruzano n. sp. from Rhamdia guatemalensis; (b) Scleroductus oaxaqueno n. sp. from Rhamdia laticauda.
Light micrographs and drawings of Scleroductus veracruzano n. sp. described from Rhamdia guatemalensis from Río La Antigua, Veracruz, Mexico: (a) hamuli complex; (b) ventral bar; (c) marginal hook; (d) dorsal bar; (e) marginal hook sickle; (f) Scleroductus lyrocleithrum marginal hook sickle; (g) male copulatory organ (MCO), * principal hook; (h) overlay of the marginal hook sickles of Scleroductus veracruzano n. sp. (solid line) and S. lyrocleithrum (dotted line).
(urn:lsid:zoobank.org:act:6F643655-5A5A-4308-A34B-73B8A38D4D91)
Type – host: Rhamdia guatemalensis (Günther, 1864) (Heptapteridae)
Type – locality: Río La Antigua, Apazapan, Veracruz, Mexico (19°19′31.49′′ N, 96°43′31.57′′ W)
Type – material: Holotype (accession no. CNHE12257), two paratypes (accession nos. CNHE12258–9) and two vouchers (accession no. CNHE12260) deposited in the Colección Nacional de Helmintos (CNHE), Universidad Nacional Autónoma de México, Mexico City
Site in host: Fins
Representative DNA sequences: ITS rDNA sequences obtained from six individuals are deposited in GenBank (accession nos. PQ733002–PQ733007), and 18S rDNA sequences obtained from two individuals (accession nos. PQ733008 and PQ733009).
Etymology: The specific epithet ‘veracruzano’ is the gentilic for the people from Veracruz in Mexico, the state where the parasite was collected.
Description
Specimens were assigned to the genus Scleroductus because they have bulbous male copulatory organs and ventral bar membranes with two prominent projections. Morphological description was based on 18 specimens, six whole worms cleared in Grey & Wess solution, and 12 proteolytically digested specimens. Body (n = 6) 422 (357–593) long; 108 (42–162) wide at uterus level. Bulbous pharynx 34 (20–55) long × 38 (25–81) wide (Figure 4a). Haptor 73 (46–98) length × 60 (45–77) width. Hamuli (anchors) robust 52 (47–56) long, 5 (4–6) wide; shaft 34 (28–36) long; point 30 (29–32) long, almost same length as hamulus shaft; proximal shaft width 8 (7–10) wide, being widest part of hook; hamulus aperture distance 18 (16–20) long; hamulus aperture angle 32° (29°–36°); hamulus root 20 (15–24) long, with rounded edges (Figure 5a). Ventral bar (superficial bar) small, 26 (23–28) wide, 14 (12–20) long; small ventral bar processes 2 (0.8–3) long; concave ventral bar median portion 10 (9–11) long, ventral bar membrane open forming two longitudinal structures 15 (13–20) long, with irregular proximal ends, almost half length of hamulus shaft (Figure 5b). Dorsal bar (deep bar) short and straight, 13 (10–16) wide, 2 (1–2) long, attachment points prominent and slender 10 (10–11) long (Figure 5d). Marginal hook 20 (17–23) long; shaft 16 (13–19) long, ending with a slight curvature at the end and attached to sickle´s heel (Figure 5c). Marginal hook sickle (hooklet) 5 (4–5) long, facing downwards toward shaft (Figure 5e). Marginal hook sickle distal width 5 (4–6) long and slim; sickle point bent towards toe; toe 3 (2–3) long, with rounded edges and facing upwards to sickle point; marginal hook base concave. Marginal hook aperture 5 (4–6) long. Heel rounded and short, with small, rounded lump at mid portion (Figures 5e, h). Marginal hook bridge short. Filament loop 16 (12–20) long, almost same length as marginal hook (Figure 5c). Male Copulatory Organ spheric, bulbous (n = 1) globular to ovoid 11 long × 10 wide, located below the pharynx; armed with 1 large spine, 5–6 small spines dissimilar in shape and size (Figure 5g). Developing embryo(s) clearly visible within adult parasite (Figure 4a).
Remarks
Scleroductus veracruzano n. sp. is the second species within the genus to be described from R. guatemalensis in Mexico; the first was S. lyrocleithrum, which it resembles. However, these species can be discriminated from each other based on the morphology of their marginal hooks (Figure 3h). The marginal hook is smaller in S. veracruzano n. sp. than in S. lyrocleithrum (4 and 6 μm, respectively). Also, in S. lyrocleithrum, the marginal hook is wider in the base of its heel (Figure 5f), while in S. veracruzano n. sp., this is narrower (Figure 5e). The marginal hook heel presents a small, rounded lump in S. veracruzano n. sp. (Figure 5e), which is absent in S. lyrocleithrum (Figure 5f). The marginal hook sickle is more robust in S. veracruzano n. sp. (wider than the marginal hook toe, when contrasting sickle at the level of the toe distal limit; Figure 5e) than in S. lyrocleithrum, where the marginal hook sickle is narrower than the toe, at the level of the toe limit (Figure 5f). Finally, the marginal hook shaft of S. veracruzano n. sp. joins the heel towards the back of the marginal hook sickle (Figure 5e), while in S. lyrocleithrum, the shaft joins the sickle close to the middle of the sickle (Figure 5f). Scleroductus veracruzano n. sp. can also be discriminated from S. yuncensi and from S. angularis based on the morphology of the hamuli and the marginal hooks. In S. veracruzano n. sp., the hamulus is almost straight and its point is almost as long as the shaft, while in both S. yuncensi and S. angularis, the hamuli are relatively curved and have shorter points. The marginal hooks also allow discrimination between Scleroductus veracruzano n. sp. and both S. yuncensi and S. angularis: the sickle base of S. veracruzano n. sp. is curved and aligned to the base and has a small, rounded lump at the heel, which is absent in both other species; the sickle base and toe are angled upwards facing the sickle point in S. yuncensi; and the marginal hook sickle in S. angularis is triangle-shaped, while in S. veracruzano n. sp., it is rounded. The ventral bar of S. veracruzano n. sp. has the characteristic shape of the genus Scleroductus (‘dumbbell-shaped; bar membrane in form of 2 longitudinal filamentous processes’ according to Jara and Cone (Reference Jara and Cone1989); ‘bar with 2 posterior ribbon-like projections that diverge and parallel anchor shafts’ according to Kritsky et al. Reference Kritsky, Boeger, Mendoza-Franco and Vianna2013); these membranes/projections have also been referred to as ‘R1 sclerites’ (Ciccheto et al. Reference Ciccheto, Razzolini, de Buron and Boeger2023). Scleroductus veracruzano n. sp. occurs in sympatry and concurrently forms mixed infections with Gyrodactylus juili García-Vásquez, Pinacho-Pinacho, Guzmán-Valdivieso, Calixto-Rojas & Rubio-Godoy, Reference García-Vásquez, Pinacho-Pinacho, Guzmán-Valdivieso, Calixto-Rojas and Rubio-Godoy2023 (García-Vásquez et al. Reference García-Vásquez, Pinacho-Pinacho, Guzmán-Valdivieso, Calixto-Rojas and Rubio-Godoy2023). Although generally similar, S. veracruzano n. sp. and G. juili can be easily discriminated, because their marginal hooks are very different (comparatively upright and thin in G. juili, curved and thick in S. veracruzano n. sp.) and the morphology of their ventral bar is markedly dissimilar: in G. juili, the ventral bar membrane is continuous, long, and triangular, while in S. veracruzano n. sp., the ventral bar membrane is split into two long, ribbon-like processes.
Description of Scleroductus oaxaqueno n. sp. Figures 4 and 6; Table 1
Light micrographs and drawings of Scleroductus oaxaqueno n. sp. described from Rhamdia laticauda from Río Chiquito, Oaxaca, Mexico: (a) hamuli complex; (b) ventral bar; (c) marginal hook; (d) dorsal bar; (e) marginal hook sickle; (f) Scleroductus lyrocleithrum marginal hook sickle; (g) male copulatory organ (MCO), * principal hook; Figures 1–7 indicate spines surrounding principal hook; (h) overlay of the marginal hook sickles of Scleroductus oaxaqueno n. sp. (solid line) and S. lyrocleithrum (dotted line).
(urn:lsid:zoobank.org:act:F616058D-3F44-477D-A130-C49295C9FEB3)
Type – host: Rhamdia laticauda (Kner, 1858) (Heptapteridae)
Type – locality: Río Chiquito, Cuicatlán, Oaxaca, Mexico (17°48′41.7′′ N, 96°57′38.7′′ W)
Type – material: Holotype (accession no. CNHE12261), two paratypes (accession nos. CNHE12262–3) and two vouchers (accession no. CNHE12264) deposited in the Colección Nacional de Helmintos (CNHE), Universidad Nacional Autónoma de México, Mexico City
Site in host: Fins
Representative DNA sequences: ITS rDNA sequences obtained from two individuals are deposited in GenBank (accession nos. PQ732998 and PQ732999), and 18S rDNA sequences obtained from two individuals (accession nos. PQ733000 and PQ733001).
Etymology: The specific epithet ‘oaxaqueno’ (‘oaxaqueño’ in Spanish) is the gentilic for the people of Oaxaca in Mexico, the state where the parasite was collected.
Description
Specimes adscribed to genus Scleroductus because of the characteristic morphology of male copulatory organ and ventral bar. Morphological description based on nine specimens, three whole worms cleared in Grey & Wess solution, and six proteolytically digested specimens. Body (n = 3) 558 (440–678) long; 150 (140–158) wide at uterus level. Pharynx 40 (31–46) long × 42 (38–48) wide (Figure 4b). Haptor 80 (72–90) length × 81 (63–92) width. Hamulus (anchor) 55 (52–59) long, 6 (5–7) wide; shaft 36 (34–39) long; point 34 (32–36) long, almost same length as hamulus shaft; proximal shaft width 9 (7–11) wide, being hamulus’ widest part; hamulus aperture distance 18 (15–21) long; hamulus aperture angle 30° (24°–37°); hamulus root 22 (17–25) long, short, narrower at end point (Figure 6a). Ventral bar (superficial bar) 15 (14–19) wide, 27 (25–28) long; ventral bar processes 2 (1–2) long, very short and rounded; ventral bar median portion 11 (9–13) long, anterior edge concave and posterior end flat, from which extends ventral bar membrane, 15 (13–16) long, almost half length of hamulus shaft, divided in two short and thin ribbon-like portions, with irregular ends (Figure 6b). Dorsal bar (deep bar) width 14 (12–18) wide, length 2 (1–2) long straight and same width throughout its length, attachment points 11 (9–13) long (Figure 6d). Marginal hook 22 (18–26) long; shaft 17 (13–20) long (Figure 6c). Marginal hook sickle (hooklet) 5 (4–6) long, facing downwards to shaft (Figure 6e). Marginal hook sickle distal width 6 (5–7) long, with irregular shaped heel. Toe 3 (3–4) long, short ending as a rounded tip; heel short and same heigh as bridge, small and round projection in mid-section. Marginal hook sickle aperture 6 (5–6) long. Filament loop 16 (13–19) long, longer than hook shaft (Figure 6c). Male copulatory organ (n = 1) globous, 26 long × 26 wide; consisting of one big principal hook, six to seven small spines, which are distributed surrounding the principal hook as: one slim and long spine and six robust triangular spines almost the same length (clockwise direction) (Figure 6g). Developing embryo(s) clearly visible within adult parasite (Figure 4b).
Remarks
Scleroductus oaxaqueno n. sp. is the first member of the genus to be described from R. laticauda, and the third species known from heptapterid catfishes in Mexico. The new species resembles the haptoral hook morphology of S. lyrocleithrum. Nonetheless, S. oaxaqueno n. sp. can be discriminated from S. lyrocleithrum based on the marginal hook morphology (Figure 6h). The marginal hook point of S. oaxaqueno n. sp. extends farther from the toe limit (Figure 6e) and is deeper than that of S. lyrocleithrum (Figure 6f). Additionally, the marginal hook heel presents a small, rounded lump in S. oaxaqueno n. sp. (Figure 6e), which is absent in S. lyrocleithrum (Figure 6f). Scleroductus oaxaqueno n. sp. can also be discriminated from S. yuncensi because the new species has straight hamuli, while these hooks are curved in the latter; marginal hooks also differ greatly, with a round, even sickle base with a small protuberance in the heel in S. oaxaqueno n. sp., while the base is comparatively straight and facing upward to the point in S. yuncensi. The haptoral hook morphology of S. oaxaqueno n. sp. and S. angularis is similar, but these species can be discriminated when comparing the marginal hooks: in S. angularis, the sickle base is relatively flat and the sickle point ends at the level of the bridge, while in S. oaxaqueno n. sp., the base is round and the marginal hook point ends below the toe base. The ventral bar membrane of S. oaxaqueno n. sp. has the characteristic shape of the genus Scleroductus, with two projections that run alongside to the hamulus shaft – these have also been referred to as ‘R1 sclerites’ (Ciccheto et al. Reference Ciccheto, Razzolini, de Buron and Boeger2023). Scleroductus oaxaqueno n. sp. occurs in sympatry and concurrently forms mixed infections with Gyrodactylus chulini García-Vásquez, Pinacho-Pinacho, Guzmán-Valdivieso, Calixto-Rojas & Rubio-Godoy, Reference García-Vásquez, Pinacho-Pinacho, Guzmán-Valdivieso, Calixto-Rojas and Rubio-Godoy2023 (García-Vásquez et al. Reference García-Vásquez, Pinacho-Pinacho, Guzmán-Valdivieso, Calixto-Rojas and Rubio-Godoy2023). Although generally similar, S. oaxaqueno n. sp. and G. chulini can be easily differentiated because their marginal hooks are very characteristic (relatively upright and thin in G. chulini, angled forward and thick in S. oaxaqueno n. sp.); and the morphology of their ventral bar is markedly contrasting: in G. chulini, the ventral bar membrane is continuous, long and V-shaped, while in S. oaxaqueno n. sp., the ventral bar membrane is split into two long, ribbon-like projections.
Phylogenetic analysis and genetic divergence
The lengths of the ITS and 18S rDNA sequences were, respectively, of 1,136 and 1,848 bp for both new species of Scleroductus. The four new 18S rDNA sequences were trimmed and added to the data set of Gyrodactylidae and Oogyrodactylidae we obtained from GenBank (Supplementary Table S1); with this, we built an alignment of 52 sequences of 1,461 bp, of which 512 were variable sites. ML and BI analyses with the 18S rDNA data set resulted in similar trees. The phylogenetic tree of the BI analyses is shown in Figure 7. The 18S rDNA fragment tree supported the erection of two new species, S. veracruzano n. sp. and S. oaxaqueno n. sp., both of which are sister species to an undescribed Scleroductus sp. (accession no. JX840361). Scleroductus spp. form a clade sister to a well-supported clade composed by members of the genera Ieredactylus, Gyrodactyloides and Laminiscus (Figure 7). ITS rDNA genetic divergence (p distances) between the two new species ranged from 3.4 to 3.6% (42 variable sites); intraspecific divergence ranged from 0 to 0.5%. The 18S rDNA interspecific genetic divergence of two new species was of 0.16% (only 4 variable sites).
Phylogenetic tree for selected species of the Gyrodactylidae and Oogyrodactylidae based on Bayesian inference of an 18S rDNA fragment, showing the position of Scleroductus veracruzano n. sp. and Scleroductus oaxaqueno n. sp. Several of the species featured are those analyzed by Boeger et al. (Reference Boeger, Kritsky, Patella and Bueno-Silva2021), whose classification into different families within Gyrodactylidea and colour shading we replicate.
Discussion
With the addition of the two new taxa described here, five species of Scleroductus are known to infect Neotropical catfishes in the Americas – plus an undescribed but molecularly characterized Scleroductus sp. recorded from R. quelen in Brazil (Kritsky et al. Reference Kritsky, Boeger, Mendoza-Franco and Vianna2013). Morphologically, the two new species share characteristic traits of the genus Scleroductus – namely, globular male copulatory organs and ventral bars whose membrane is split into two long projections. Despite their general resemblance, the known species of Scleroductus can be discriminated based on the morphology of their hamuli and marginal hooks – a phenomenon also observed for several species of Gyrodactylus (e.g., García-Vásquez et al. Reference García-Vásquez, Razo-Mendivil and Rubio-Godoy2015).
Based on the phylogenetic hypothesis we present, S. veracruzano n. sp. and S. oaxaqueno n. sp. collected from Rhamdia spp. in Mexico are closely related to each other, and both appear as sister species to Scleroductus sp. from R. quelen in Brazil (GenBank JX840361); however, this position could be an artifact, as this sample from Brazil is currently the only available sequence for members of Scleroductus, so the phylogeny we present could change as soon as other taxa are characterized molecularly. The ITS rDNA sequences we obtained from the new taxa support the erection of different species, as genetic divergence between S. veracruzano n. sp. and S. oaxaqueno n. sp. was >3%, a value considered significant enough to discriminate between species of Gyrodactylus (Ziętara and Lumme Reference Ziętara and Lumme2002). However, we cannot propose any phylogenetic hypothesis for Scleroductus based on this marker, as no ITS sequences are available in GenBank for members of this genus. Nonetheless, our analyses based on 18S rDNA confirm the placement of Scleroductus sp. within the Gyrodactylidae, a monophyletic family sister to Oogyrodactylidae (Boeger et al. Reference Boeger, Kritsky, Patella and Bueno-Silva2021). In addition, our data support the hypothesis of one clade within Gyrodactylidae being composed by species of the genera Scleroductus+Ieredactylus+Laminiscus+Gyrodactyloides, which is similar to hypotheses previously proposed by Boeger et al. (Reference Boeger, Kritsky, Patella and Bueno-Silva2021) and Ciccheto et al. (Reference Ciccheto, Razzolini, de Buron and Boeger2023).
Scleroductus sp. has been recorded extensively in South America, infecting several species of Neotropical catfishes (Siluriformes) throughout the continent (Chemes et al. Reference Chemes, Takemoto and Sottini2008; Dias et al. Reference Dias, Alves, Da Silva, Abdallah and De Azevedo2017; dos Santos et al. Reference dos Santos, de CastroLemos-Pita and Brasil-Sato2007; Ferrari-Hoeinghaus et al. Reference Ferrari-Hoeinghaus, Takemoto, Oliveira, Makrakis and Baumgartner2006; Gião et al. Reference Gião, Pelegrini, De Azevedo and Abdallah2020; Gutiérrez and Martorelli Reference Gutiérrez and Martorelli1999; Jara and Cone Reference Jara and Cone1989; Kritsky et al. Reference Kritsky, Boeger, Mendoza-Franco and Vianna2013; Negreiros et al. Reference Negreiros, Tavares-Dias and Pereira2019; Negrelli et al. Reference Negrelli, Iannacone, Abdallah and De Azevedo2021; Sabas annd Brasil-Sato 2014). Considering that two parasite species (S. lyrocleithrum and S. veracruzano n. sp.) infect the same host species (R. guatemalensis) in Mexico, we assume that several undescribed parasite species are associated to the very speciose Neotropical catfishes distributed in the Americas. However, in the absence of molecular data for these widely distributed gyrodactylids, it is impossible to assess their diversity and infer their evolutionary history – which is a clear knowledge shortfall (Mendoza-Palmero et al. Reference Mendoza-Palmero, Blasco-Costa and Scholz2015; Rubio-Godoy and Pérez-Ponce de León Reference Rubio-Godoy and de León G2023). Nonetheless, it can be hypothesized that species of Scleroductus collected in South America will be closely related to other Neotropical taxa – in particular, to Scleroductus sp. collected from R. quelen in Brazil (GenBank JX840361). It can further be hypothesized that different clades of monogenean parasites infect catfishes in South America and in North America. This latter hypothesis is based on the results of this work (Figure 5) and supported by the observation that species of Gyrodactylus infecting Rhamdia spp. in Mexico are sister species to each other and form a sister clade to gyrodactylids infecting R. quelen in Brazil (García-Vásquez et al. Reference García-Vásquez, Pinacho-Pinacho, Guzmán-Valdivieso, Calixto-Rojas and Rubio-Godoy2023). A similar pattern is observed in characid fishes, where gyrodactylids infecting fish hosts in Mexico are closely related to each other and contained within a sister clade to their congeners infecting characid fishes in Brazil (Pozos-Carré et al. Reference Pozos-Carré, Pinacho-Pinacho, Calixto-Rojas, Caspeta-Mandujano, Ramírez-Herrejón, García-Vásquez, Barrios-Gutiérrez, Guzmán-Valdivieso and Rubio-Godoy2025).
The occurrence in North America (Mexico) of various monogenean parasite species with Neotropical affinities (Choudhury et al. Reference Choudhury, García-Varela and Pérez-Ponce De León2017; García-Prieto et al. Reference García-Prieto, Dáttilo, Rubio-Godoy and Pérez-Ponce de León2022) has been highlighted as support for the hypothesis that following the biogeographical event known as the Great American Biotic Interchange (GABI)((Bacon et al. Reference Bacon, Molnar, Antonelli, Crawford, Montes and Vallejo-Pareja2016)), several Neotropical freshwater fish species migrated north via the emerging Isthmus of Panama – and that their associated parasites co-migrated with them (Choudhury et al. Reference Choudhury, García-Varela and Pérez-Ponce De León2017) and diversified, presumably as their fish hosts colonized and adapted to new environments (Pozos-Carré et al. Reference Pozos-Carré, Pinacho-Pinacho, Calixto-Rojas, Caspeta-Mandujano, Ramírez-Herrejón, García-Vásquez, Barrios-Gutiérrez, Guzmán-Valdivieso and Rubio-Godoy2025). The present work further strengthens this hypothesis.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X25000264.
Acknowledgements
We thank Luis García-Prieto, Colección Nacional de Helmintos, Universidad Nacional Autónoma de México, Mexico, for the loan of type specimens.
Financial support
This research was partially funded by Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT), Mexico (M.R.G., grant numbers CB-168306, CF-2023-G-362), (C.D.P.P., grant number CF-2023-G-95). We thank CONAHCYT, Mexico, for scholarships granted to A.G.V. (CVU 392202), M.C.R. (CVU 717160), and D.A.P.C. (CVU 1210106).
Competing interests
The author(s) declare none.
Ethical standard
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.
