Introduction
The Neotropical region has the most diverse freshwater fish fauna in the world, with more than 6,000 known species (Albert et al. Reference Albert, Tagliacollo and Dagosta2020), being dominated by fishes from the orders Siluriformes, Characiformes, and Gymnotiformes, which constitute approximately 77% of the species (Justo et al. Reference Justo, Martins and Cohen2023). The Characiformes is considered the second most dominant order of fishes in this region (Albert et al. Reference Albert, Tagliacollo and Dagosta2020; Fricke et al. Reference Fricke, Eschmeyer and Fong2021; Tonella et al. Reference Tonella and Lucena2022), and its representatives live exclusively in freshwater habitats (Nelson et al. Reference Nelson, Grande and Wilson2016), possessing a wide taxonomic, morphological, and behavioural richness (Javonillo et al. Reference Javonillo, Malabarba, Weitzman and Burns2010).
Tetragonopterus Cuvier, 1816 is a taxonomically complex genus of the Characidae, currently represented by 15 species (Froese and Pauly, Reference Froese and Pauly2025). Tetragonopterus argenteus Cuvier, 1816 occurs in freshwater ecosystems of Northeastern Brazil and is an economically important species used for human consumption (Silva et al. Reference Silva, Melo, Oliveira and Benine2016; Muniz et al. Reference Muniz, Flamini, Kantek, Lázaro, De Souza and Junior2019). Carvalho et al. (Reference Carvalho, Sousa, Diniz and Yamada2022) investigated the diversity of metazoan parasites in T. argenteus from the Jaguaribe River basin, recording 10 distinct taxa, among which only a single monopisthocotylan species was found parasitizing the nasal cavities of its hosts. Monopisthocotylans are the most diverse helminth parasites of freshwater fishes and one of the best-studied taxa in the Neotropical region (Luque et al. Reference Luque, Pereira, Alves, Oliva and Timi2017). However, most monopisthocotylan descriptions are based on morphological data and do not provide molecular characterization of the described species (Diniz et al. Reference Diniz, Sousa, Yamada and Yamada2025; Franceschini et al. Reference Franceschini, Zago, Müller, Francisco, Takemoto and Silva2018; Poulin et al. Reference Poulin, Presswell and Jorge2020; Silva et al. Reference Silva, Silva and Yamada2021; Yamada et al. Reference Yamada, Diniz, Sousa, Yamada and Tavares-Dias2024a). On the other hand, recent studies have used molecular markers for more precise species identification, as well as for carrying out phylogenetic analyses (Ebert et al. Reference Ebert, Osaki-Pereira, De León and Silva2024; Hasuike et al. Reference Hasuike, Scorsim, Arjona, Bellay, Oliveira and Takemoto2024; Osaki-Pereira et al. Reference Osaki-Pereira, Narciso, Vieira, Müller, Ebert and Silva2023; Santos-Neto and Domingues, Reference Santos-Neto and Domingues2023, Reference Santos-Neto and Domingues2024; Yamada et al. Reference Yamada, Müller, Zago, Yamada, Ebert, Franceschini and Silva2023; Zago et al. Reference Zago, Franceschini, Abdallah, Müller, Azevedo and Silva2021).
To date, the known diversity of monopisthocotylan parasites of the nasal cavities of Neotropical freshwater fishes is restricted to 26 species belonging to seven genera, as follow: Characithecium Mendoza-Franco, Reina, and Torchin, Reference Mendoza-Franco, Reina and Torchin2009 (two species); Diaphorocleidus Jogunoori, Kritsky, and Venkatanarasaiah, Reference Jogunoori, Kritsky and Venkatanarasaiah2004 (two species); Pavanelliella Kritsky and Boeger, 1998 (five species); Rhinoxenus Kritsky, Boeger, and Thatcher, 1988 (eleven species); Rhinonastes Kritsky, Thatcher, and Boeger, 1988 (two species); Telethecium Kritsky, Van Every, and Boeger, 1996 (three species); and Urocleidoides Mizelle and Price, 1964 (one species) (Santos-Neto et al. Reference Santos-Neto, Paixão and Domingues2024). Of this total, 22 species have been reported in fishes from Brazilian freshwater ecosystems.
Herein, we describe a new species of Diaphorocleidus parasitizing the nasal cavities of T. argenteus, from the Lima Campos weir, municipality of Icó, Ceará state, Brazil, supported by morphological and molecular data (COI mtDNA and LSU rDNA). Furthermore, the ribosomal LSU and the mitochondrial COI gene region were used to analyze the phylogenetic position of the new species in relation to other Neotropical dactylogyrids.
Material and methods
Host sampling and parasitological procedures
Fifty-four specimens of T. argenteus were collected with the aid of cast nets, between September 2019 and March 2020, in the Lima Campos weir (6°22′59.99″S, 38°58′0.01″W), Salgado River sub-basin, municipality of Icó, state of Ceará, Brazil. Collection of fish specimens was authorized under the Permanent License for Collection of Zoological Material (SISBIO n° 61328-1) and all animal procedures were carried out in full compliance with the Ethics Committee for Animal Experimentation (CEUA/protocols n° 00165/2018.1) of the Regional University of Cariri (URCA), municipality of Crato, state of Ceará, Brazil.
After capture, fish specimens were individually stored in plastic bags, frozen, and taken to the laboratory. At the time of necropsy, an incision was made in the host’s nostrils using scissors, followed by washing the nasal cavities with tap water, and the contents were stored in Petri dishes. All monopisthocotylans found were collected and placed in vials containing 70% ethanol. Parasites were mounted on slides with Gray and Wess’s medium to study the sclerotized structures (Kritsky et al. Reference Kritsky, Thatcher and Boeger1986). Prevalence, mean intensity of infestation, and range were calculated according to Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997).
In March 2024, a new collection was carried out at the same location. Forty recently deceased hosts were examined in situ, and only three monopisthocotylans specimens were recovered. In the first step, the parasites found were placed in a drop of 0.9% saline solution and identified under a microscope. Two specimens were placed in 99% molecular-grade ethanol. Subsequently, one specimen was sent to LAPAS (Laboratório de Parasitologia de Animais Silvestres) of São Paulo State University (UNESP) for molecular analysis, and the other specimen was mounted in Gray and Wess’ medium as a molecular representative – Paragenophore (Pleijel et al. Reference Pleijel, Jondelius, Norlinder, Nygren, Oxelman, Schander, Sundberg and Thollesson2008). Specimens were registered in SISGEN (Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado) under the registration number (A05E891). One specimen was fixed in 5% formalin, stained with Gomori’s trichrome, and mounted in Gray and Wess’s medium for analyses of the internal organs (Kritsky et al. Reference Kritsky, Leiby and Kayton1978).
Morphology and morphometry of the parasites were analyzed using an optical microscope equipped with a computerized system for phase-contrast image analysis (Leica ICC50W). All measurements are in micrometres and represent straight-line distances between extreme points expressed as the mean followed by the range, and the number (n) of structures measured in parentheses. Measurements of the sclerotized parts of the haptor (total length [d1] and total width [d2]) are represented in Figure 1, and the total length of the male copulatory organ (MCO) was measured using the freehand tool. Direction of the MCO (counterclockwise vs. clockwise) followed Kritsky et al. (Reference Kritsky, Boeger and Thatcher1985). Numbering and distribution of hook pairs followed Mizelle and Price (Reference Mizelle and Price1963). Illustrations were made with the aid of a drawing tube mounted on a Leica DM750 microscope with phase-contrast optics and vectorized using Inkscape 1.4 software.
Diaphorocleidus radius n. sp. parasite of the nasal cavities of Tetragonopterus argenteus Cuvier, 1816 (Characiformes: Characidae), in the Lima Campos weir, municipality of Icó, Ceará state, Brazil. (a) Whole mount (composite, ventral view); (b) copulatory complex (ventral view); (c) vagina; (d) ventral anchor; (e) dorsal anchor; (f) hooks; (g) egg; (h) ventral bar; (i) dorsal bar. Scale bars: (a) = 200 μm; (b–g) = 50 μm; (h–i) = 20 μm.
Type specimens and paragenophore of monopisthocotylans were deposited in the Collection of the Oswaldo Cruz Institute (CHIOC), Rio de Janeiro state, Brazil. Paratypes were deposited in the Helminthological Collection of the Institute of Biosciences of Botucatu (CHIBB) of the São Paulo State University (UNESP), municipality of Botucatu, São Paulo state, Brazil.
DNA extraction, amplification, and sequencing
In a second step of molecular analyses, the monopisthocotylan specimen was mounted on a slide with a drop of ethanol, covered with a coverslip, and examined under a microscope. Once identified, the specimen, preserved in absolute ethanol, was used for DNA extraction. The total genomic DNA was isolated using the DNeasy® Blood and Tissue Kit (Qiagen), following the manufacturer’s protocol. The extraction yielded a final volume of 30 μL, which was used to sequence fragments. Gene fragments of the LSU rDNA and COI mtDNA were amplified using conventional polymerase chain reaction (PCR). Each reaction mixture contained 3 μL of extracted DNA, 12.5 μL of 2× MyFi™ Mix (Bioline, Taunton, MA, USA), 1 μL of each primer, and 7.5 μL of nuclease-free H₂O, totalling 25 μL. For the LSU rDNA gene, amplification and sequencing were carried out using the primers 382F (5′-AGC TGG TGG AGT CAA GCT TC-3′) and 1289R (5′-TGC TCA CGT TTG ACG ATC GA-3′) (Yamada et al. Reference Yamada, Müller, Zago, Yamada, Ebert, Franceschini and Silva2023). PCR conditions included an initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 2 min, with a final extension at 72°C for 10 min (Yamada et al. Reference Yamada, Müller, Zago, Yamada, Ebert, Franceschini and Silva2023).
For the COI mtDNA gene, the primers COI Mono 5 (5′-TAA TWG GTG GKT TTG GTA A-3′) and COI Mono 3 (5′-TAA TGC ATM GGA AAA AAA CA-3′) (Plaisance et al. Reference Plaisance, Rousset, Morand and Littlewood2008) were used. Thermocycling protocol consisted of an initial denaturation at 94°C for 3 min, followed by 40 cycles of 94°C for 30 s, 47°C for 30 s, and 72°C for 1 min and 30 s, ending with a final extension at 72°C for 7 min.
PCR products were visualized on a 1% agarose gel stained with GelRed™. PCR amplicons were then purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, Inc.), following the manufacturer’s instructions. Sequencing was performed directly on the purified PCR products using a BigDye v.3.1 Terminator Cycle Sequencing Ready Reaction kit on an ABI3730xl Genetic Analyzer (Applied Biosystems). Sequences were edited and assembled using Sequencher v.5.2 (Gene Codes).
Phylogenetic inferences
Newly obtained LSU rDNA and COI mtDNA sequences were individually aligned with other Dactylogyridae sequences retrieved from GenBank (Table 1). For the LSU rDNA dataset, 76 sequences of taxonomically related species were retrieved from GenBank and aligned, resulting in a final alignment of 1,040 bp after trimming ambiguous regions. Sequences from Murraytrema pricei Caballero, Bravo, and Grocott, 1955, Pseudorhabdosynochus epinepheli (Yamaguti, 1938), and Pseudorhabdosynochus lantauensis (Beverley-Burton and Suriano, 1981) were used as outgroups.
List of monopisthocotylans included in the phylogenetic analyses, with details of the host, locality, and GenBank accession numbers of sequences from the large subunit ribosomal (LSU rDNA) and mitochondrial cytochrome oxidase subunit I (COI mtDNA) genes. New sequences obtained for the present study are in bold
For the COI mtDNA dataset, 32 sequences of taxonomically related species were retrieved from GenBank and aligned, resulting in a final alignment of 638 bp after trimming ambiguous regions. Sequence of Acanthocotyle gurgesiella Ñacari, Sepúlveda, Escribano, and Oliva, 2017, was used as an outgroup. Alignments were constructed using Geneious v.7.1.3, employing the MUSCLE algorithm with default parameters. Sequences were trimmed to match the length of the shortest sequence in each dataset.
Prior to phylogenetic analyses, the optimal evolutionary model for both the LSU rDNA and COI mtDNA datasets was determined using jModelTest 2 (Posada Reference Posada2008), accessed via the CIPRES online platform (Miller et al. Reference Miller, Pfeiffer and Schwartz2010). Model selection was based on the Akaike Information Criterion (AIC), with the GTR + I + G Model identified as the best fit for both datasets.
Phylogenetic relationships were inferred using Bayesian Inference (BI) and Maximum Likelihood (ML) methods. For BI, analyses were performed using MrBayes (Ronquist and Huelsenbeck, Reference Ronquist and Huelsenbeck2003) on the CIPRES platform. Markov Chain Monte Carlo (MCMC) simulations were run for 106 generations, with trees sampled every 100 generations. The initial 250,000 generations were discarded as burn-in, and only nodes with posterior probabilities exceeding 0.95 were considered strongly supported. ML analyses were conducted using RAxML v8 (Guindon and Gascuel, Reference Guindon and Gascuel2003) on the CIPRES platform, with 1000 bootstrap replicates. Bootstrap values above 70% were regarded as well-supported.
Pairwise genetic divergence was calculated using the Tamura–Nei model in MEGA11 (Tamura et al. Reference Tamura, Stecher and Kumar2021) for both LSU rDNA and COI mtDNA datasets. Rate variation among sites was modelled using a gamma distribution, and all ambiguous positions were excluded for each sequence pair. The resulting BI and ML tree was visualized using FigTree v.1.4.4 (Rambaut Reference Rambaut2020) and further refined using CorelDraw X8 for graphical representation.
Results
Taxonomic Summary
Class Monopisthocotyla Brabec, Salomaki, Scholz, and Kuchta, 2023
Order Dactylogyridea Bychowsky, 1937
Dactylogyridae Bychowsky, 1933
Diaphorocleidus Jogunoori, Kritsky, and Venkatanarasaiah, Reference Jogunoori, Kritsky and Venkatanarasaiah2004
Diaphorocleidus radius n. sp. Carvalho, Diniz, Sousa, and Yamada (Figures 1 and 2)
Photomicrographs of the sclerotized pieces of the Diaphorocleidus radius n. sp. from the nasal cavities of Tetragonopterus argenteus Cuvier, 1816 (Characiforme: Characidae), in the Lima Campos weir, municipality of Icó, Ceará state, Brazil. (a) Copulatory complex (ventral view), (b) haptor (showing the lightning-shaped of the ventral anchor tip). Scale bars: 50 μm.
Syn: Ancistrohaptor sp. of Carvalho et al. (Reference Carvalho, Sousa, Diniz and Yamada2022)
(urn:lsid:zoobank.org:pub:6CD5FB0C-6040-4EE6-ABAE-DF5341807F04)
Type host: Tetragonopterus argenteus Cuvier, 1816 (Characiformes: Characidae).
Type locality: Lima Campos weir (6°22′59.99″S, 38°58′0.01″W), Salgado River sub-basin, municipality of Icó, state of Ceará, Brazil.
Site of infestation: Nasal cavities.
Infestation parameters: Prevalence: 57% (31 of 54 analyzed hosts); Mean intensity of infestation and range: 1,70 ± 0,16 (1–5).
Type material: Holotype CHIOC 40879, paratypes CHIOC 40880 - 40885 and CHIBB 934L - 939L, paragenophore CHIOC 40886.
Molecular sequence data: LSU rDNA (GenBank accession number PX574983; 885 bp) and COI mtDNA (GenBank accession number PX525833; 638 bp).
Etymology: The specific epithet is from Latin-derived (radius = lightning, bolt) and refers to the morphology/shape of the ventral anchor tip.
Description: [Based on 24 specimens: one stained with Gomori’s trichrome, and 23 mounted in Gray and Wess’s medium] Body fusiform, 880 (652–1,152, n = 20) long, 278 (183–354, n = 20) wide. Cephalic region wide; cephalic lobes poorly developed, three bilateral pairs of cephalic organs, cephalic glands not observed. Two pairs of eyes. Accessory granules in anterior region of trunk. Pharynx spherical. Male copulatory organ 357 (302–402, n = 10) long, coiled tube with 2½ up to 3.0 counterclockwise rings; base with sclerotized margin and proximal flap; proximal ring diameter 26 (18–34, n = 24). Accessory piece 49 (43–59, n = 24) long, sclerotized, bifurcate, pincer-shaped, composed of two subunits articulated. Articulation process connecting the base of the MCO with accessory piece present. Haptor subhexagonal; dissimilar anchors with well-developed roots. Ventral anchor 45 (37–53, n = 24) long, 74 (63–84, n = 24) wide, well-developed superficial root, deep root with tip ornamentation, recurved shaft, elongated, and lightning-shaped tip. Dorsal anchor 30 (27–33, n = 21) long, 16 (12–20, n = 21) wide, elongated superficial root, short deep root, recurved shaft with prominence in proximal portion and short tip. Ventral bar 35 (32–38, n = 22) long, 11 (8–13, n = 22) wide, robust, plate-shaped, truncated in lateral ends. Dorsal bar 27 (24–31, n = 21) long, 6 (5–8, n = 21) wide, smaller than the ventral bar, moderately U-shaped, rounded in lateral ends. Hooks 19 (10–26, n = 24) long, with protruding thumb, delicate shaft and point; shank composed of two subunits; expanded proximal subunit; filament hook (FH) 1/4 of the shank. Vaginal aperture submarginal, vagina sinistral, forming a sclerotized sinuous tube, emptying to seminal receptacle. Seminal receptacle midventral, anterior to germarium. Germarium ventral to testis, 249 (188–299, n = 6) long, 87 (78–101, n = 5) wide. Seminal vesicle elongated; single prostatic reservoir. Egg 84 (n = 2) long, 62 (n = 2) wide. Testis, oviduct, ootype, and uterus not observed. Vitelline follicles densely dispersed throughout trunk except in region of reproductive organs.
Remarks: Diaphorocleidus radius n. sp. was allocated to the genus Diaphorocleidus by having a tubular MCO with counterclockwise rings; an accessory piece composed of two subunits articulated; overlapping gonads; and hooks of similar shape, with a shaft composed of two subunits, proximal subunit dilated. The new species resembles Diaphorocleidus affinis (Mizelle, Kritsky, and Crane, 1968), Diaphorocleidus forcipiformis Silva, Meneses, Cohen, and Justos, Reference Silva, Cohen, Ottoni, Viana, Meneses and Justo2024, Diaphorocleidus forficata Santos-Neto, Paixão, and Domingues, 2024, Diaphorocleidus munimensis Silva, Meneses, Cohen, and Justos, Reference Silva, Cohen, Ottoni, Viana, Meneses and Justo2024 , Diaphorocleidus petrosusi Mendoza-Franco, Aguirre-Macedo, and Vidal-Martinez, 2007, and Diaphorocleidus sclerocolpus Santos-Neto, Costa, Soares, and Domingues, 2018 in the morphology of the accessory piece, all by having a bifurcated accessory piece (pincer-shaped), composed of two subunits articulated. The new species resembles Diaphorocleidus magnus Zago, Franceschini, Abdallah, Müller, Azevedo, and Silva, Reference Zago, Franceschini, Abdallah, Müller, Azevedo and Silva2021, by having an articulation process connecting the base of the MCO with accessory piece. However, Diaphorocleidus radius n. sp. can be easily distinguished from all congeners by the morphology of the ventral anchor with well-developed superficial root, ornamented deep root, recurved blade, elongated and lightning-shaped tip; and ventral bar robust, plate-shaped, truncated in lateral ends.
Phylogenetic relationships of Diaphorocleidus radius n. sp. within the Dactylogyridae
The phylogenetic analysis of LSU rDNA sequences, conducted using both Bayesian Inference (BI) and Maximum Likelihood (ML) methods, revealed the position of Diaphorocleidus radius n. sp. within the Dactylogyridae. A partial sequence of the LSU rDNA gene from one specimen of Diaphorocleidus radius n. sp. (885 bp long; GenBank accession number PX574983) was obtained. Both ML and BI analyses of the LSU rDNA dataset yielded similar tree topologies, with moderate to high support values across most nodes. Diaphorocleidus radius n. sp. forming a distinct clade associated with acestrorhamphid hosts. Phylogenetic analyses based on partial LSU rDNA sequences revealed that Diaphorocleidus armillatus (the type species of the genus) and D. forficata formed an independent clade closely related to species of Cacatuocotyle. However, this clade was weakly supported (Figure 3). The remaining species of Diaphorocleidus were recovered in a different lineage, together with other dactylogyrid genera. This topology indicates that Diaphorocleidus, as currently defined, does not represent a monophyletic group.
Bayesian topology based on LSU rDNA sequences showing the position of Diaphorocleidus radius n. sp. (highlighted in bold). Nodes showing topological incongruence were represented by a dash. The branch length scale bar indicates the number of substitutions per site.
For the COI mtDNA, one sequence of Diaphorocleidus radius n. sp. (638 bp long; GenBank accession number PX525833) was obtained. Both ML and BI analyses of the COI mtDNA dataset yielded similar tree topologies, with most branches displaying low support values. Four internal nodes showed differences in support between the ML and BI analyses, with ML presenting slightly lower bootstrap values for some branches while BI maintained high posterior probabilities. Despite these variations, the position of Diaphorocleidus radius n. sp. was consistently well-supported across both analyses (Figure 4).
Bayesian topology based on COI mtDNA sequences showing the position of Diaphorocleidus radius n. sp. (highlighted in bold). Nodes showing topological incongruence were represented by a dash. The branch length scale bar indicates the number of substitutions per site.
The COI mtDNA analysis revealed that Diaphorocleidus radius n. sp. is most closely related to D. petrosusi, forming a sister clade, although this relationship was not significantly supported (pp = 0.74; bs = 67). Together, these two species form a clade with D. magnus, D. neotropicalis Zago, Franceschini, Abdallah, Muller, Azevedo, and Silva, Reference Zago, Franceschini, Abdallah, Müller, Azevedo and Silva2021, and Urocleidoides strombicirrus (Price and Bussing, 1967), which may reflect a recent divergence, but further data are needed to confirm their relationships. For instance, species of Urocleidoides, including U. nataliapasternakae Santos-Neto and Domingues, 2023, U. uncinus Zago, Yamada, Yamada, Franceschini, Bongiovani, and Silva, Reference Zago, Yamada, Yamada, Franceschini, Bongiovani and Silva2020, and U. curvocuspidis Ebert, Osaki-Pereira, Pérez-Ponce de León, and Silva, Reference Ebert, Osaki-Pereira, De León and Silva2024, are recovered as a closely related group but remain phylogenetically distinct from the Diaphorocleidus clade. Similarly, R. paranaensis Rossin and Timi, 2019, and species of Jainus Mizelle, Kritsky and Crane, 1968, such as J. piava Karling, Bellay, Takemoto, and Pavanelli, 2011, and J. beccus Yamada, Muller, Zago, Yamada, Ebert, Franceschini, and Silva, Reference Yamada, Müller, Zago, Yamada, Ebert, Franceschini and Silva2023, form their independent lineages when compared to Diaphorocleidus radius n. sp. and its congeners.
The genetic divergence estimates based on the p-distance analyses provide strong support for the recognition of Diaphorocleidus radius n. sp. as a distinct taxonomic entity within the genus Diaphorocleidus (Supplementary material S1). The LSU rDNA dataset revealed a substantial genetic distance of 30% between Diaphorocleidus radius n. sp. and D. forficata, and 23% from D. armillatus. These values greatly exceed the commonly interspecific divergence threshold for monopistocotylans, which typically ranges from 10% to 25% for LSU rDNA sequences, suggesting that Diaphorocleidus radius n. sp. represents a genetically distinct taxon within the currently unresolved relationships of the genus.
Interestingly, the divergence between Diaphorocleidus radius n. sp. and species of the congeneric genus Cacatuocotyle Boeger, Domingues, and Kritsky, 1996, reached up to 18%, which is lower than the observed divergence from some other Diaphorocleidus species. This pattern may reflect a complex evolutionary history within these dactylogyrid lineages, suggesting that certain species currently assigned to Diaphorocleidus are highly divergent and may warrant further taxonomic scrutiny. Nevertheless, the genetic distance from Cacatuocotyle spp. remains above the thresholds typically used to distinguish genera within Dactylogyridae, and the phylogenetic position of Diaphorocleidus radius n. sp. supports its placement within Diaphorocleidus.
Across other congeners, Diaphorocleidus radius n. sp. exhibited average genetic distances ranging from 14% to 23%. These results align with previous studies highlighting high intraspecific homogeneity and pronounced interspecific divergence in COI mtDNA sequences among dactylogyrids (Supplementary material S2). Therefore, the genetic evidence supports the interpretation that Diaphorocleidus radius n. sp. represents a novel and well-differentiated species within Diaphorocleidus, corroborating the morphological distinctions observed in the present study.
Discussion
Species of Diaphorocleidus are characterized primarily by the presence of counterclockwise rings in the MCO, an accessory piece that is non-articulated with the MCO, a sinistral vagina, and hooks with proximal shank dilatation (Jogunoori et al. Reference Jogunoori, Kritsky and Venkatanarasaiah2004). More recently, Zago et al. (Reference Zago, Franceschini, Abdallah, Müller, Azevedo and Silva2021) amended the diagnosis to include: a copulatory complex comprising either an articulated or unarticulated MCO and accessory piece; an articulation process connecting the base of the MCO to the accessory piece, which may be present or absent; variable shapes of the accessory piece; and ventral anchors larger than dorsal ones. The morphological characteristics described herein support the recognition of Diaphorocleidus radius n. sp. as a new species within the genus.
Currently, 15 species are considered valid in Diaphorocleidus. These species have been reported from Iguanodectidae (three species), Acestrorhamphidae (six), Acestrorhynchidae (two), Bryconidae (three), Hemiodontidae (one) (see Table 2). The new species described in the present study from the nasal cavities of T. argenteus represents the first record of the genus on a host from the Characidae. Among them, only Diaphorocleidus radius n. sp. and D. forficata are known to exclusively parasitize the nasal cavities.
List of species of Diaphorocleidus described from the Neotropical region
Abbreviations: G: gills; N: nasal cavities; S: body surface; SI: site of infestation.
According to Zago et al. (Reference Zago, Franceschini, Abdallah, Müller, Azevedo and Silva2021), the presence of Diaphorocleidus spp. in different characiform families may be linked to the phylogenetic relationships among their hosts. Braga et al. (Reference Braga, Razzolini and Boeger2015), in exploring factors influencing parasite sharing among Neotropical freshwater fishes, proposed that phylogenetic contiguity within Characiformes likely facilitated host-switching events via a ‘stepping stone’ process, as the diversification of this fish lineage occurred within the Neotropics. This hypothesis is further supported by Mendoza-Franco et al. (Reference Mendoza-Franco, Caspeta-Mandujano and Ramírez-Martínez2019), Zago et al. (Reference Zago, Franceschini, Abdallah, Müller, Azevedo and Silva2021), and Santos-Neto et al. (Reference Santos-Neto, Paixão and Domingues2024). Thus, phylogenetic proximity among Neotropical Characiformes may promote the sharing of Diaphorocleidus species across families within this order.
Molecular phylogenetic analyses based on LSU rDNA and COI mtDNA markers provide important insights into the systematic placement of Diaphorocleidus radius n. sp. In the LSU rDNA topology, Diaphorocleidus radius n. sp. appears in a clade with other Diaphorocleidus species, particularly those parasitizing hosts in the family Acestrorhamphidae, although this clade is not well supported. Diaphorocleidus forficata and D. armillatus are recovered as sister species, yet the relationships among Diaphorocleidus spp. and between Diaphorocleidus and Cacatuocotyle remain poorly resolved due to low nodal support. Although the limited support cautions against definitive conclusions, this topology fails to recover the genus as monophyletic, raising questions about its current taxonomic boundaries and emphasizing the need for further investigation. This concern is further underscored by the relatively high genetic divergence (30%) observed in the LSU rDNA between Diaphorocleidus radius n. sp. and D. forficata, despite their morphological similarities, particularly in the morphology of the accessory piece, and their shared microhabitat (nasal cavities). However, since the LSU rDNA is a highly conserved marker among invertebrates, such values should be interpreted with caution. Rather than indicating a considerable evolutionary separation, this divergence may reflect the limited resolution of this gene, emphasizing the need to rely on node support and additional molecular markers to better assess the relationships between these lineages.
The COI mtDNA phylogeny, although generally exhibiting low branch support, complements these results by reinforcing the genetic distinctiveness of Diaphorocleidus radius n. sp. In this analysis, Diaphorocleidus radius sp. n. clusters with D. petrosusi, D. magnus, D. neotropicalis, and U. strombicirrus, the latter currently considered incertae sedis by Kritsky et al. (Reference Kritsky, Thatcher and Boeger1986). The inclusion of U. strombicirrus within the same clade as Diaphorocleidus species supports the suggestion by Alda et al. (Reference Alda, Mendoza-Franco, Hanson-Regan, Reina, Bermingham and Torchin2025) of a potential reassignment, given its close relationship to species of Diaphorocleidus. Rodríguez-González et al. (Reference Rodríguez-González, Mendoza-Franco, Caspeta-Mandujano and Salgado-Maldonado2025) conducted a morphological analysis of anchor structures, which was inconclusive in assigning U. strombicirrus and therefore did not confirm its generic status, further reinforcing the need for integrative approaches to clarify its phylogenetic placement. Therefore, although it is most likely that this species could be transferred to Diaphorocleidus, no morphological characteristics have yet been identified that clearly justify such a taxonomic decision.
Additional genetic divergence estimates further strengthen the case for taxonomic reconsideration. LSU rDNA sequences show 23% divergence between Diaphorocleidus radius n. sp. and D. armillatus, the type species of the genus. These values exceed interspecific thresholds and, interestingly, approach the level of divergence observed between Diaphorocleidus radius n. sp. and members of Cacatuocotyle, a phylogenetically related genus, with divergence reaching up to 18%. This indicates that some Diaphorocleidus lineages may be as genetically distinct from one another as they are from entirely separate genera. Although the morphological characteristics of Diaphorocleidus radius n. sp. remain consistent with the broadened diagnosis of the genus proposed by Zago et al. (Reference Zago, Franceschini, Abdallah, Müller, Azevedo and Silva2021), the molecular data support its inclusion within Diaphorocleidus and suggest that the current boundaries of the genus may require further evaluation once additional sequences become available.
Position of D. armillatus adds another layer of complexity. While this species forms a supported clade with D. forficata in the LSU rDNA tree, this grouping has weak support relative to its sister clade, which includes species of Cacatuocotyle. This weak support limits confidence in the current phylogenetic placement of D. armillatus, and by extension, the stability of the genus Diaphorocleidus as a whole. To resolve this uncertainty, further molecular data from a broader taxon sampling, including more representatives of Cacatuocotyle and Diaphorocleidus, are essential to test the monophyly and clarify the evolutionary relationships within this lineage.
Genetic divergence estimates based on COI mtDNA sequences provide robust evidence supporting the recognition of Diaphorocleidus radius n. sp. as a distinct taxonomic entity within the genus Diaphorocleidus. The genetic distances between Diaphorocleidus radius n. sp. and other congeneric species ranged from 15% to 18%, values that fall within the upper limits of interspecific divergence observed for dactylogyrid using this mitochondrial marker. Notably, the average intergeneric divergence for COI mtDNA among related genera such as Urocleidoides, Rhinoxenus, and Jainus is approximately 22%, further underscoring the significance of the observed genetic distinctiveness of Diaphorocleidus radius n. sp. Interestingly, the divergence between Diaphorocleidus radius n. sp. and representatives of the genus Cacatuocotyle reached up to 18%, which is equal to or, in some cases, lower than the divergence observed with some Diaphorocleidus species.
In addition to its phylogenetic implications, Diaphorocleidus radius n. sp. represents the first Diaphorocleidus species recorded from a characid host, and also the third known instance of a species from this genus parasitizing the nasal cavities of Neotropical fishes. This microhabitat among Monopisthocotyla may reflect a case of specialization or an adaptive strategy that allows exploitation of less competitive niches.
Taken together, these findings, including the description of Diaphorocleidus radius n. sp., enhance our understanding of the phylogenetic placement of Diaphorocleidus species and highlight the importance of integrative taxonomic approaches. By combining morphological and molecular data, researchers can more accurately elucidate the evolutionary history of dactylogyrid monopisthocolylans. The discovery of Diaphorocleidus radius n. sp., a genetically distinct yet morphologically consistent, illustrates the complexity of adaptive radiation in these parasites, particularly within the rich and dynamic host environment offered by Neotropical Characiformes.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X2510103X.
Acknowledgements
The study received partial support through research grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)—for the productivity research grant awarded to R.J.S. (311635/2021-0) and F.H.Y. (174814/2023-2), and for the research grant to P.O.F.Y. (151170/2022-3) and F.H.Y. (304502/2022-7); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)—M.N.M.C. (88887.520213/2020-00), W.B.B.S. (88887.704478/2022-00), and M.M.O.P. (88887.955939/2024-00); and Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP)—P.O.F.Y. (FPD-0213-00301.01.01/23) and M.F.B.G.D. (BMD-0008-02422.01.09/23).
Author contribution
Writing the manuscript and methodology, M.N.M.C., M.F.B.G.D., P.O.F.Y., and W.B.B.S.; vectorized-illustration, W.B.B.S. and P.O.F.Y.; morphometrical analysis, M.F.B.G.D.; molecular data and phylogenetic inferences, M.M.O.P. and R.J.S.; review-editing, F.H.Y. All authors have read and approved the published manuscript version.
Competing interests
The authors declare they have no conflicts of interest.
Ethical approval
All animal procedures were performed in full compliance with the Ethics Committee for Animal Experimentation (CEUA/URCA #00165/2018.1).
