Introduction
The order Siluriformes constitutes an exceptionally diverse group of fishes and stands as one of the oldest in the world (Kappas et al. Reference Kappas, Vittas, Pantzartzi, Drosopoulou and Scouras2016). This order encompasses the family Ariidae, comprising 42 genera and 157 species. Members of this family inhabit marine and aquatic environments across the globe (Eschmeyer 2023; Marceniuk et al. Reference Marceniuk, Oliveira and Ferraris2024).
To date, 78 species of monopisthocotyleans have been found to parasitise Ariidae worldwide (Soares et al. Reference Soares, Adriano, Domingues and Balbuena2023). Among these species, 69 belong to Dactylogyridae, including representatives from genera such as Chauhanellus Bychowsky & Nagibina, Reference Bychowsky and Nagibina1969, Hamatopeduncularia Yamaguti, 1953, Neotetraonchus Bravo-Hollis, Reference Bravo-Hollis1968, and Susanlimocotyle Soares, Domingues & Adriano, Reference Soares, Domingues and Adriano2021. Additionally, there are seven species classified under the Neocalceostomatidae, encompassing genera such as Fridericianella Brandes, 1894, Neocalceostoma Tripathi, 1959, Neocalceostomoides Kritsky, Mizelle & Bilqees, Reference Kritsky, Mizelle and Bilqees1978, and Thysanotohaptor Kritsky, Shameem, Kumari, & Krishnaveni, Reference Kritsky, Shameem, Kumari and Krishnaveni2012.
The Peruvian sea catfish, Galeichthys peruvianus Lütken, 1874 (Siluriformes: Ariidae), is a demersal fish endemic to Peru (Kulongowski Reference Kulongowski2010; Froese and Pauly Reference Froese and Pauly2023; Fricke et al. Reference Fricke, Eschmeyer and van der Laan2023) that inhabits coastal waters (Kailola and Bussing Reference Kailola, Bussing, Fischer, Krupp, Schneider, Sommer, Carpenter and Niem1995; Froese and Pauly Reference Froese and Pauly2023). Hitherto, surveys on monogeneans from G. peruvianus had been restricted to one record: the dactylogyrid Hamatopeduncularia sp. (Iannacone and Luque Reference Iannacone and Luque1993; Luque et al. Reference Luque, Cruces, Chero, Paschoal, Alves, Da Silva, Sánchez and Iannacone2016).
During a study on gill ectoparasites on marine fishes from Peru, two new species of Neotetraonchus Bravo-Hollis, Reference Bravo-Hollis1968, N. celsomanueli sp. nov. and N. peruvianus sp. nov., were found infecting G. peruvianus. Herein, the new species are figured and described.
Materials and methods
Specimen collection and morphological analyses
Specimens of the Peruvian sea catfish were captured by the artisanal fishery during January 2018 and February 2019 off the coastal zone of Puerto Pizarro, Tumbes region (3°29’S, 80°24’W), northern Peru. Fish were immediately dissected upon capture, and the gill arches were removed and placed in vials containing heated sea water (60°C). Each vial underwent vigorous shaking, after which formalin was added to obtain a 4% solution. In the laboratory, the contents of each vial were examined under a dissecting microscope, and monopisthocotyleans were removed from the gill filaments or sediment using small probes. Some monopisthocotyleans were directly fixed in 70% ethanol and subsequently preserved in 90% ethanol until further processing. The anterior and posterior parts of these specimens were cut and used for morphological identification, whereas the middle sections were used for molecular procedures. Some specimens were stained with Gomori’s trichrome (Humason Reference Humason1979), clarified in clove oil, and mounted in Canada balsam for observing internal anatomy. Other specimens were mounted in Hoyer’s medium (Humason Reference Humason1979) for studying sclerotised structures. Examination and photography of specimens were conducted using a compound NikonTM Eclipse SI photomicroscope (Tokio, Japon) equipped with phase contrast light microscopy, and drawings were made with the aid of a drawing tube. Measurements are presented in micrometers (μm), representing straight-line distances between extreme points of the structures measured, and are expressed as the range followed by the mean and number (n) of structures measured in parentheses. Dimensions of the haptoral and copulatory sclerites follow the proposal by Kritsky et al. (Reference Kritsky, Mendoza-Franco, Bullard and Vidal-Martínez2009). Body length represents the length of the body proper with the haptor. Numbering of haptoral-hook pairs followed the system of Mizelle (Reference Mizelle1936) and Mizelle and Price (Reference Mizelle and Price1963). Anatomical terms for Neotetraonchus follow Kritsky et al. (Reference Kritsky, Mendoza-Franco, Bullard and Vidal-Martínez2009). The direction of the coil (clockwise vs. counterclockwise) of the male copulatory organ (MCO) follows Kritsky et al. (Reference Kritsky, Boeger and Thatcher1985). Fishes were identified according to the keys of Peruvian marine fishes by Chirichigno and Vélez (Reference Chirichigno and Vélez1998) and Chirichigno and Cornejo (Reference Chirichigno and Cornejo2001). The type-material of the species studied in the present paper was deposited in the Helminthological Collection of the Museum of Natural History at the San Marcos University (MUSM), Peru.
Molecular characterisation and phylogenetic analysis
The DNA samples were isolated from a single specimen of a new monopisthocotylean species using the Qiagen QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocol. This DNA underwent polymerase chain reaction (PCR) amplification using primers C1 (5′ -ACCCG CTGAA TTTAA GCAT- 3′) and D2 (5′ -TGGTC CGTGT TTCAA GAC- 3′) (Littlewood et al. Reference Littlewood, Curini-Galletti and Herniou2000; Lockyer et al. Reference Lockyer, Olson and Littlewood2003; Olson et al. Reference Olson, Cribb, Tkach, Bray and DTJ2003) to amplify and sequence the partial 28S rDNA gene. The thermocycling profile applied was per Mendoza-Palmero et al. (Reference Mendoza-Palmero, Blasco-Costa and Scholz2015). Samples were sequenced utilising an ABI 3730 DNA analyser from the RPT01A subunit for DNA sequencing, available at the Technological Platforms Network of the Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
Sequences were edited and contigs were assembled using ProSeq 2.9 beta (Filatov Reference Filatov2002). The National Center for Biotechnology Information sequence database (henceforth ‘GenBank’) was searched for similar sequences using Basic Local Alignment Search Tool (BLAST) (Altschul et al. Reference Altschul, Gish, Miller, Myers and Lipman1990). Sequences generated in this study were aligned with selected sequences obtained from GenBank, using Clustal W software (Larkin et al. Reference Larkin, Blackshields, Brown, Chenna, McGettigan, McWilliam, Valentin, Wallace, Wilm and Lopez2007) (Supplementary Table S1). Pseudomurraytrematoides pricei (DQ157672), Pseudorhabdosynochus lantauensis (AY553624), and Pseudorhabdosynochus epinepheli (AY553622) (Diplectanidae) were used as the outgroup for the 28S rDNA. The aligned dataset was analysed with JModelTest2 software (Santorum et al. Reference Santorum, Darriba, Taboada and Posada2014), which compares different models of DNA substitution in a hierarchical hypothesis-testing framework to select a base substitution model that best fits the data for each gene. The best model found was TVM + I + G, selected with Bayesian information criterion (BIC). The model parameters were as follows: assumed nucleotide frequencies A = 0.1967, C = 0.1870, G = 0.2689, and T = 0.3474; substitution rate matrix with A-C substitution = 1.2088, A-G = 5.2010, A-T = 2.6301, C-G = 0.6526, C-T = 5.2010, G-T = 1.000; p-inv distribution and gamma distribution with shape parameters = 0.1800 and 1.0240, respectively. Next, the best model was implemented in MrBayes 3.2.7a (Huelsenbeck and Ronquist Reference Huelsenbeck and Ronquist2001) for Bayesian inference analysis (BI). For the BI analysis, unique random starting trees were used in Metropolis-coupled Markov chain Monte Carlo (MCMC) (Ronquist, et al. Reference Ronquist, Huelsenbeck and Teslenko2011). The analysis was performed for a total of 5,000,000 generations. Visual inspection of log-likelihood scores against generation time indicated that the log-likelihood values reached a stable equilibrium before 100,000 generations. Thus, a burn-in of 1000 samples was conducted; every 100th tree was sampled from the MCMC analysis, obtaining a total of 100,000 trees, and tree topology represented the 50% majority rule consensus trees. Support for nodes in the BI tree topology was obtained by posterior probability. The phylogenetic analyses were conducted using the Cyberinfrastructure for Phylogenetic Research Science Gateway V. 3.3 platform (http://www.phylo.org/ (Miller et al. Reference Miller, Pfeiffer and Schwartz2011). Phylogenetic trees were visualised and edited in Figtree 1.4.4 (Rambaut Reference Rambaut2009). Pairwise genetic distances between the sequences of 28S rDNA were calculated in MEGA X (Kumar et al. Reference Kumar, Stecher, Li, Knyaz and Tamura2018) using the Kimura 2-Parameter model (Kimura Reference Kimura1980).
Results
Taxonomic account
Class. Monopisthocotylea Odhner, 1912
Order. Dactylogyridea Bychowsky, 1937; Dactylogyridae Bychowsky, 1933; Neotetraonchus Bravo-Hollis, Reference Bravo-Hollis1968
Neotetraonchus celsomanueli sp. nov. Cruces, Chero, Ñacari & Luque
https://zoobank.org/act:985C5E83-D43D-4263-9563-5F7E104DF719
Figure 1. Neotetraonchus celsomanueli sp. nov. from the Peruvian sea catfish Galeichthys peruvianus. (A) Whole specimens (ventral view); (B) Ventral anchor; (C) Dorsal anchor; (D) Onchium; (E) Ventral bar; (F) Dorsal bar; (G) Hook pair 1; (H) Hook pair 2; (I) Hook pair 3; (J) Hook pair 4; (K) Hook pair 5; (L) Hook pair 6; (M) Hook pair 7; (N) Male copulatory organ.
Type host. Galeichthys peruvianus Lütken, 1874 (Siluriformes, Ariidae), Peruvian sea catfish.
Site in host. Gill filaments.
Type locality. Puerto Pizarro (3°29’S, 80°24’W), Tumbes, Peru, South America.
Deposited material. Holotype: MUSM 5402; paratypes: MUSM 5403a-j.
Molecular sequence data. The 28S rDNA (823 bp), obtained from one specimen, was deposited in GenBank (accession numbers OR886655).
Etymology. This species is named in honor of Celso Manuel Cruces Torres, father of the first author.
Description. Based on 5 specimens mounted in Hoyer medium and 6 specimens stained with Gomori’s trichrome: Body elongate, fusiform, slender, 985–1308 (1198; n = 6) long; greatest width 112–158 (134; n = 6), usually at level of prostatic reservoir. Tegument thin, surface smooth. Cephalic region moderately broad; lateral lobes poorly developed; 3 bilateral pairs of conspicuous head organs; bilateral pair of cephalic glands at postpharyngeal level. Four eye-spots, equidistant, members of anterior pair smaller than those of posterior pair; chromatic granules absent. Pharynx spherical, muscle-glandular, 51–71 (62; n = 6) in diameter; esophagus short; intestinal bifurcation postpharyngeal; intestinal ceca not confluent posteriorly to gonads, lacking diverticula. Peduncle broad, elongate. Haptor differentiated from body proper, almost subhexagonal, with bilateral lobes, 126–187 (156; n = 6) long; 170–219 (192; n = 6) wide; well-developed secretory gland lying on peduncle, plate-shaped. Onchium ventral in haptor, 77–109 (93; n = 11) long, forked-shaped, with long posteriorly directed bilateral arms. Anchors dissimilar, lacking well-defined roots. Ventral anchor with fine conspicuous inner filament, 82–88 (85; n = 4) long; evenly curved shaft; doubly recurved point. Dorsal anchor 81–83 (82; n = 4) long, with large rootless base; delicate shaft and point. Ventral extrinsic adductor muscle well developed, broadly inverted U-shaped. Ventral bar 154–177 (163; n = 4) long, tiara-shaped, with medium anteromedial projection, bilobated lateral ends. Dorsal bar 85–104 (92; n = 4) long, rod-shaped, elongated, with two developed anterolateral protuberances, expanded lateral ends. Fourteen hooks, dissimilar in size and shape; hook pairs 1–4 and 6–7 having shanks comprised of 2 variably expanded subunits; pair 1 with a grossly expanded proximal subunit of shank, near onchium; proximal subunit of pairs 2–4 and 6 variable in length and shape; hook pair 5 with uniform shank, comprised of single subunit, lying along shaft and point of ventral anchor; filamentous hook loop extends beyond level of junction of both subunits in pairs 1, 2, 4, and 7, extend at level of junction of both subunits in pairs 3 and 6, and about shank length in pair 5; hook pair 1, 59–63 (61; n = 4) long; hook pair 2, 32–33 (33; n = 4) long; hook pair 3, 23–25 (24; n = 4) long; hook pair 4, 22–25 (23; n = 4) long; hook pair 5, 15–16 (16; n = 4) long; hook pair 6, 26–31 (28; n = 4) long; hook pair 7, 101–109 (103; n = 4) long. MCO 250–316 (282; n = 11) long, tubular, with ring-shaped base and proximally expanded shaft; shaft with T-shaped tip and counterclockwise ring at distal end of proximal expansion; accessory piece ribbed and expanded proximally, with worm-shaped termination. Testis ovate, intercecal, dorsal to ovary, 90–123 (101; n = 6) long, 31–40 (36; n = 6) wide; vas deferens looping left intestinal cecum, dilating to form sinuous seminal vesicle in left side of trunk, anterolateral to MCO; single elongate ovate prostatic reservoir, dorsal to MCO. Ovary 90–119 (102; n = 6) long, 29–48 (40; n = 6) wide; ovate, overlapping anterior portion of testis. Vaginal aperture dextrolateral, prominent; vaginal vestibule large, well developed, sclerotised and bowl-shaped proximally; vaginal duct narrow, short, running posteriorly to join large subspherical seminal receptacle lying near body mid-line and overlying anterior end of ovary. Oviduct, oötype, and uterus not observed. Vitelline follicles dense throughout trunk, lateral fields of follicles confluent anterior to MCO, anterior to ovary and posterior to testis. Egg not observed.
Remarks
Neotetraonchus celsomanueli sp. nov. differs from all congeners mainly by having a tubular MCO with a ring-shaped base and a hammer-shaped distal end, and by having an accessory piece ribbed and expanded proximally with worm-shaped termination. Members of the new species slightly resemble N. bychowskyi Bravo-Hollis, Reference Bravo-Hollis1968 from the Gulf of Mexico in the morphology of their anchors and ventral bars. However, the new species differs from N. bychowskyi in the morphology of the accessory piece, which is ribbed and attached to the medial region of the shaft of the MCO, as opposed to a non-ribbed rod accessory piece in N. bychowskyi (see Figure 2 of Kritsky et al. Reference Kritsky, Mendoza-Franco, Bullard and Vidal-Martínez2009). In addition, both species can be differentiated by the morphology of the MCO (T-shaped tip in Neotetraonchus celsomanueli sp. nov. vs. flared tip in N. bychowskyi) and by the morphology of the onchium (plate subtrapezoidal with two anterolateral knob and large anteromedial projection in the new species vs. plate subquadrangular with short anteromedial projection in N. bychowskyi). Neotetraonchus celsomanueli sp. nov. also resemble N. bravohollisae Paperna, 1977 from the north Atlantic Ocean in the morphology of the MCO (expanded distally). However, N. celsomanueli sp. nov. differ from N. bravohollisae in the morphology of the accessory piece of the MCO copulatory complex (ribbed and expanded proximally with worm-shaped termination in N. celsomanueli sp. nov. vs. slender rod with sickle-shaped termination in N. bravohollisae), onchium (subtrapezoidal plate in the new species vs. with subquadrangular plate in N. bravohollisae), and morphology of the ventral bar (tiara-shaped with medium anteromedial projection in N. celsomanueli sp. nov. vs. an inverted U-shape with a short anteromedial projection in N. bravohollisae).
Figure 2. Neotetraonchus peruvianus sp. nov. from the Peruvian sea catfish Galeichthys peruvianus. (A) Whole specimens (ventral view); (B) Ventral anchor; (C) Dorsal anchor; (D) Onchium; (E) Ventral bar; (F) Dorsal bar; (G) Hook pair 1; (H) Hook pair 2: (I) Hook pair 3; (J) Hook pair 4; (K) Hook pair 5; (L) Hook pair 6; (M) Hook pair 7; (N) Male copulatory organ.
Neotetraonchus peruvianus sp. nov. Cruces, Chero, Ñacari & Luque
https://zoobank.org/act:4BA46D7C-17F4-49FA-94E6-F1B15A9361AA
Type host. Galeichthys peruvianus Lütken, 1874 (Siluriformes, Ariidae), Peruvian sea catfish.
Site in host. Gill filaments.
Type locality. Puerto Pizarro (3°29’S, 80°24’W), Tumbes, Peru, South America.
Deposited material. Holotype: MUSM 5404; paratypes: MUSM 5405a-g.
Molecular sequence data. It was not possible to obtain the molecular sequences of this species.
Etymology. The specific epithet of the new species refers to the country where it was discovered.
Description. Based on 4 specimens mounted in Hoyer medium and 4 specimens stained with Gomori’s trichrome: Body elongate, fusiform, slender to robust, 847–1094 (976; n = 4) long; greatest width 137–164 (152; n = 4), usually at level of prostatic reservoir. Tegument thin, surface smooth. Cephalic region moderately broad; lateral lobes poorly developed; 3 bilateral pairs of conspicuous head organs; bilateral pair of cephalic glands at pre and postpharyngeal level. Four, equidistant, members of anterior pair smaller than those of posterior pair; chromatic granules absent. Pharynx spherical, eye spots muscle-glandular, 53–69 (60; n = 4) in diameter; esophagus short; intestinal bifurcation postpharyngeal; intestinal ceca not confluent posteriorly to gonads, lacking diverticula. Peduncle broad, elongate. Haptor differentiated from body proper, almost subrectangular, with bilateral lobes, 95–124 (109; n = 4) long; 136–164 (149; n = 4) wide; well-developed secretory gland lying on peduncle, elongate. Onchium ventral in haptor, 44–58 (53; n = 8) long, jellyfish-shaped, with long posteriorly directed bilateral arms. Anchors dissimilar, lacking well-defined roots. Ventral anchor with fine conspicuous inner filament, 49–55 (52; n = 4) long; evenly curved shaft; doubly recurved point. Dorsal anchor 46–51 (49; n = 4) long, with large rootless base; delicate shaft and point. Ventral extrinsic adductor muscle well developed, broadly inverted U-shaped. Ventral bar 106–113 (111; n = 4) long, broadly inverted V-shaped, with broad anteromedial projection, bilobulated lateral ends. Dorsal bar 73–76 (74; n = 4) long, bandeau-shaped, elongated, with two developed anterolateral projections, expanded lateral ends. Fourteen hooks, dissimilar; hook pairs 1–4 and 6–7 having shanks comprised of 2 variably expanded subunits; pair 1 with grossly expanded proximal subunit of shank, near onchium; proximal subunit of pairs 2–4 and 6 variable in length and shape; hook pair 5 with undilated shank, comprised of single subunit, lying along shaft and point of ventral anchor; filamentous hook loop extends beyond level of junction of both subunits in pairs 1, 2, 4, and 7, extend at level of junction of both subunits in pairs 3 and 6, and about shank length in pair 5; hook pair 1, 40–45 (42; n = 4) long; hook pair 2, 26–31 (29; n = 4) long; hook pair 3, 23–26 (25; n = 4) long; hook pair 4, 23–27 (25; n = 4) long; hook pair 5, 16–17 (16; n = 3) long; hook pair 6, 31–36 (33; n = 4) long; hook pair 7, 62–66 (65; n = 4) long. MCO 172–226 (194; n = 8) long, tubular, with ring-shaped base and proximally expanded shaft; shaft with sledgehammer-shaped distal end, with counterclockwise ring at distal end of proximal expansion; accessory piece ribbed and expanded proximally, with claw-shaped distal end. Testis ovate, intercecal, dorsal to ovary, 57–79 (70; n = 4) long, 38–53 (46; n = 4) wide; vas deferens looping left intestinal cecum, dilating to form sinuous seminal vesicle in left side of trunk, anterolateral to MCO; single well-developed, ovate prostatic reservoir, dorsal to MCO. Ovary 72–86 (79; n = 4) long, 41–48 (45; n = 4) wide; ovate, overlapping anterior portion of testis. Vaginal aperture dextrolateral, prominent; vaginal vestibule large, well developed, sclerotised, and funnel-shaped proximally; vaginal duct narrow, short, running posteriorly to join large subspherical seminal receptacle lying near body mid-line and overlying anterior end of ovary. Oviduct, oötype, and uterus not observed. Vitelline follicles dense throughout trunk, lateral fields of follicles confluent anterior to MCO and posterior to testis. Egg not observed.
Remarks
Neotetraonchus peruvianus sp. nov. is unique among its congeners by its MCO, which has a sledgehammer-shaped distal end and an accessory piece with claw-shaped distal end. In addition, N. peruvianus sp. nov. is typified by having a jellyfish-shaped onchium.
Phylogenetic relationships
In this study, a partial 28S rDNA sequence of 823 base pairs (bp) was obtained from Neotetraonchus celsomanueli sp. nov (OR886655). BLAST analyses revealed no significant match with any other available Monopisthocotylea sequences in GenBank. The closest similarities were observed with with Hamatopeduncularia thalassini (KF676631), Thysanotohaptor rex (KF676634), and Chauhanellus auriculatum (MN108169) of ariid catfishes, showing a range of similarity between 82.61–83.11%. (See supplementary table S1). The genetic divergences between Neotetraonchus celsomanueli sp. nov. and Thysanotohaptor rex, as well as Neocalceostomoides spinivaginalis, varied by 16% (84 bp). Between Hamatopeduncularia spp. and Chauhanellus spp., the divergence ranged from 17 to 23% (83–112 bp). (See supplementary Table S2).
The phylogenetic analysis was performed based on the partial 28S rDNA gene of dactylogyrid species exclusively from Siluriformes, including our newly identified species. The analyses revealed two distinct clades with robust support in Bayesian inference (BI) analyses (Figure 3). Clade A exhibited strong support and further divided into two well-supported clades: clade A1, which subdivided into A1′ and A1′′. Clade A1′ comprised monopisthocotylean parasites of diadromous and marine catfishes (Ariidae) such as Hamatopeduncularia spp., Chauhanellus spp., Thysanotohaptor rex, Neocalceostomoides spinivaginalis, and Neotetraonchus celsomanueli sp. nov. Clade A1′′ included Schilbetrema sp. from freshwater catfishes (Schilbeidae), closely related to dactylogyrid species of Ariid catfishes. Clade A2 encompassed species parasitising freshwater catfishes (Pimelodidae, Doradidae, Loricariidae, and Heptapteridae) (Figure 3).
Figure 3. Phylogenetic tree based on 28S region for Neotetraonchus celsomanueli sp. nov. (Dactylogyridae) inferred by Bayesian inference (BI) to show their relationships with other species of Dactylogyridae infecting ariid hosts. Numbers along branches indicate the bootstrap values obtained from the posterior probability of BI. GenBank accession numbers are shown, and the scale bar indicates the number of nucleotide mutations per site.
Clade B also displayed strong support and was divided into two subclades (B1 and B2), including species parasitising freshwater catfish. Clade B1 comprised dactylogyrid species exclusively parasitsing loricariids, such as Heteropriapulus spp., Trinigyrus spp., and Unilatus unilatus. Clade B2 encompassed species like Ameloblastella spp. from Pimelodidae and Heptapteridae, Vancleaveus janauacaensis from Doradidae, and Unibarra paranoplatensis from Pimelodidae (Figure 3).
Discussion
The present survey revealed the presence of two new species of Neotetraonchus, N. celsomanueli sp. nov. and N. peruvianus sp. nov., found on marine catfish Galeichthys peruvianus, a fish common to local markets in central and northern Peru but little studied in relation to its helminth parasites.
Neotetraonchus was proposed and included in the family Neotetraonchidae by Bravo-Hollis (Reference Bravo-Hollis1968) due to specific characteristics such as possessing 16 larval hooks (eight pairs), a sacciform (non-bifurcating) gut, and a sclerotised tube-like copulatory complex. However, a subsequent revision by Kritsky et al. (Reference Kritsky, Mendoza-Franco, Bullard and Vidal-Martínez2009) reevaluated the genus, suggesting that the Neotetraonchus species actually features 14 hooks (seven pairs) and a gut composed of two caeca, aligning more with the typical features of the Dactylogyridae. To date, four valid Neotetraonchus species have been reported infesting marine ariid catfishes (Kritsky et al. Reference Kritsky, Mendoza-Franco, Bullard and Vidal-Martínez2009, Soares et al. Reference Soares, Adriano, Domingues and Balbuena2023).
The BI phylogenetic analysis (Figure 3), employing partial 28S rDNA sequences, shows the phylogenetic position of the genus Neotetraonchus within Clade A1’, grouping with members of the Neocalceostomidae (Thysanotohaptor and Neocalceostomoides) and Dactylogyridae (Chaunhanellus and Hamatopeduncularia). However, this suggests a possible confusion in the taxonomic position of the neocalceostomids, which is likely closely related to the Dactylogyridae. The Neocalceostomatidae has not been definitively established or universally accepted, yet the family is tentatively recognised to include species with an adult disc-shaped haptor armed with a pair of ventral anchors and 14 pairs of hooks (10 marginal and 4 ventral) (Boeger and Kritsky Reference Boeger, Kritsky, Littlewood and Bray2001; Kritsky et al. Reference Kritsky, Shameem, Kumari and Krishnaveni2012).
Traditionally, the Dactylogyridae comprised the subfamilies Ancylodiscoidinae Gusev, 1961 and Ancyrocephalinae Bychowsky, 1937 (Boeger and Kritsky Reference Boeger and Kritsky1993). The BI phylogenetic analysis supports the possibility that the families Neotetraonchidae Bravo-Hollis, Reference Bravo-Hollis1968 (now Dactylogyridae) and Neocalceostomidae Lim, 1995 could potentially be reclassified as subfamilies within the Dactylogyridae, as Neotetraonchinae and Neocalceostominae, respectively. However, determining the precise subfamily within the Dactylogyridae, from a molecular perspective, requires further sequence data from closely related taxa.
Neotetraonchus celsomanueli sp. nov. and N. peruvianus sp. nov. are the first species of the genus found infecting a Galeichthys species inhabiting Peruvian waters. Based on the present study and other recent reports on the monopisthocotyleans infecting fishes from northern Peru, we assume that the actual diversity of monopisthocotyleans in Peru and in the Southeast Pacific is still poorly known, and description of many new taxa, especially on poorly studied hosts, can be anticipated.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X24000208.
Acknowledgements
The authors are grateful to the following people who helped with collection of fishes in Peru: Ivette Cuellar, Eva G. Huancachoque, Alexander Reyes, Milagros Carrillo, and Cynthia E. Rodríguez, all from the National University Federico Villarreal. We also want to thank Arnaldo Maldonado Júnior for his kindness in lending us his laboratory facilities.
Financial support
C.L. Cruces was supported by a student fellowship from the Coordenação de Aperfeiçoamento de Pessoal do Ensino Superior, Brazil - Finance Code 001. J.L. Luque was supported by a researcher fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil.
Competing interest
The authors declare that they have no conflicts of interest.
Ethical standard
All applicable institutional, national, and international guidelines for the care and use of animals were followed.
Data availability
All data that support the findings of this study are available in the main text and supplementary material.
