Introduction
The family Clinostomidae Luhe, 1901 comprises digenetic trematodes, which in their adult stage are predominantly found in the oral cavity and esophagus of birds and reptiles (Kanev et al. Reference Kanev, Radev and Fried2002). These parasites exhibit a complex life cycle with gastropods serving as their first intermediate hosts and fish and amphibians as their second intermediate hosts (Pérez-Ponce De Léon et al. 2016). In their metacercariae stage, these trematodes infect a wide variety of fish hosts, having been found in at least 12 families of freshwater fish: Cichlidae, Percidae, Centrarchidae, Symbranchidae, Eleotridae, Heptapteridae, Profundulidae, Poecilidae, Goodeidae, Characidae, Cyprinidae, and Catostomidae (Acosta et al. Reference Acosta, Caffara, Fioravanti, Utsunomia, Zago, Franceschini and Silva2016; Briosio-Aguilar et al. Reference Briosio-Aguilar, Pinto, Rodríguez-Santiago, López-García, García-Varela and De León2018; Caffara et al. Reference Caffara, Locke, Gustinelli, Marcogliese and Fioravanti2011, Reference Caffara, Davidovich, Falk, Smirnov, Ofek, Cummings, Gustinelli and Fioravanti2014, Reference Caffara, Locke, Echi, Halajian, Benini, Luus Powell, Tavakol and Fioravanti2017; Davies et al. Reference Davies, Ostrowski de Núñez, Ramallo and Nieva2016; Dias et al. Reference Dias, Eiras, Machado, Souza and Pavanelli2003; Gustinelli et al. Reference Gustinelli, Caffara, Florio, Otachi, Wathuta and Fioravanti2010; Locke et al. Reference Locke, Caffara, Marcogliese and Fioravanti2015; Morais et al. Reference Morais, Varella, Fernandes and Malta2011; Pérez-Ponce de León et al. Reference Pérez-Ponce de León, García Prieto and Mendoza-Garfías2007, Reference Pérez-Ponce de León, García-Varela, Pinacho-Pinacho, Sereno-Uribe and Poulin2016; Pinto et al. Reference Pinto, Caffara, Fioravanti and Melo2015; Sereno-Uribe et al. Reference Sereno-Uribe, Pinacho-Pinacho, García-Varela and Pérez-Ponce de León2013, Reference Sereno-Uribe, García-Varela, Pinacho-Pinacho and Pérez-Ponce de León2018; Szidat Reference Szidat1969).
Based on morphological and molecular descriptions, 23 valid species of Clinostomum Leidy, 1856 have been identified thus far, and yet at least eight candidate new species have been registered through DNA sequences but have not yet been described (Briosio-Aguilar et al. Reference Briosio-Aguilar, Pinto, Rodríguez-Santiago, López-García, García-Varela and De León2018; Goméz-Ruíz and Lenis Reference Gómez-Ruíz and Lenis2024; Tavares-Dias et al Reference Tavares-Dias, Silva and Florentino2021); however, the taxonomic validity of species within Clinostomum has been a contentious issue due to the absence of significant morphological characters that differentiate the valid species from each other, along with the wide distribution of the genus, which is considered cosmopolitan, and its complex life cycle (Caffara et al. Reference Caffara, Davidovich, Falk, Smirnov, Ofek, Cummings, Gustinelli and Fioravanti2014; Locke et al. Reference Locke, Caffara, Marcogliese and Fioravanti2015, Reference Locke, Caffara, Barčák, Sonko, Tedesco, Fioravanti and Li2019; Matthews and Cribb Reference Matthews and Cribb1998; Rosser et al. Reference Rosser, Alberson, Woodyard, Cunningham and Pote2017; Sereno-Uribe et al. Reference Sereno-Uribe, García-Varela, Pinacho-Pinacho and Pérez-Ponce de León2018).
Presently, molecular and morphological analyses have revealed a high biodiversity of lineages and a large database of mitochondrial and nuclear DNA sequences of Clinostomum are available, facilitating the correct identification of species, thereby effectively estimating the diversity of this genus (Acosta et al. Reference Acosta, Caffara, Fioravanti, Utsunomia, Zago, Franceschini and Silva2016; Briosio-Aguilar et al. Reference Briosio-Aguilar, Pinto, Rodríguez-Santiago, López-García, García-Varela and De León2018; Caffara et al. Reference Caffara, Locke, Gustinelli, Marcogliese and Fioravanti2011, Reference Caffara, Davidovich, Falk, Smirnov, Ofek, Cummings, Gustinelli and Fioravanti2014, Reference Caffara, Locke, Echi, Halajian, Benini, Luus Powell, Tavakol and Fioravanti2017; Gustinelli et al. Reference Gustinelli, Caffara, Florio, Otachi, Wathuta and Fioravanti2010; Locke et al. Reference Locke, Caffara, Marcogliese and Fioravanti2015, Reference Locke, Caffara, Barčák, Sonko, Tedesco, Fioravanti and Li2019; Pérez-Ponce de León et al. Reference Pérez-Ponce de León, García Prieto and Mendoza-Garfías2007, Reference Pérez-Ponce de León, García-Varela, Pinacho-Pinacho, Sereno-Uribe and Poulin2016; Pinto et al. Reference Pinto, Caffara, Fioravanti and Melo2015; Sereno-Uribe et al. Reference Sereno-Uribe, Pinacho-Pinacho, García-Varela and Pérez-Ponce de León2013; Sereno-Uribe et al. Reference Sereno-Uribe, García-Varela, Pinacho-Pinacho and Pérez-Ponce de León2018).
During a biodiversity survey of fish parasites in the Paraná river basin, we found Clinostomidae larvae in the musculature of Serrasalmus spilopleura Kner 1858 (Characiformes: Serrasalmidae) and Callichthys callichthys (Linnaeus 1758) (Siluriformes: Callichthyidae) from southeast Brazil, and we conducted morphological and molecular analysis with the aim to characterize these metacercariae since many fish parasites larvae can be zoonotic. Additionally, we analyzed the phylogenetic position of these larvae for the first time.
Material and methods
Study area and sampling design
The Ibitinga reservoir is situated within the Tietê River Basin, a part of the larger Paraná River Basin, located in São Paulo state, Brazil. Spanning approximately 12,300 hectares, the reservoir boasts an average depth of 8.6 meters (Vieira et al. Reference Vieira, Ferreira, Castro and Rocha2002). It is primarily fed by two main tributaries – namely, the Jacaré-Pepira River (22°30’S; 47°55’W) and the Jacaré-Guaçu River. During sampling near the confluence of the Jacaré-Pepira and Tietê Rivers in Ibitinga city (21°54’46’’S; 48°53’14’’W), five specimens of S. spilopleura were collected.
In contrast, the Jurumirim dam is located on the Paranapanema River, also within the Paraná River Basin (Agostinho et al. Reference Agostinho, Vazzoler, Thomaz, Tundisi, Bicudo and Matsumura-Tundisi1995). Serving as the primary reservoir in a cascade system for downstream river regulation (Henry and Nogueira Reference Henry, Nogueira and Henry1999), it is situated in São Paulo state (23°12’17’’ S; 49°13’19’’ W). Twenty specimens of C. callichthys were gathered from the Jurumirim reservoir.
Following collection, specimens were transported in plastic bags to an ichthyoparasitology laboratory for analysis. Muscle tissue from the host fish was filleted, and cysts were carefully extracted and examined under a stereomicroscope. Sections were stained using Mayer’s carmine alum, mounted on slides, and coverslipped with Canada balsam to facilitate detailed visualization of internal structures (Eiras et al. Reference Eiras, Takemoto and Pavanelli2006). Indexes of parasite prevalence, intensity, and abundance were calculated according to the method described by Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997).
Fish hosts
Serrasalmus spilopleura (Kner 1858), commonly known as piranha or pirambeba, belongs to the Characidae family within the order Characiformes. This species is widely distributed across South America (Braga Reference Braga1976; Vazzoler and Menezes Reference Vazzoler and Menezes1992), predominantly inhabiting lentic environments such as lakes, rivers, and reservoirs (Corredor Reference Corredor2004; Saint-Paul et al. Reference Saint-Paul, Zuanon, Villacorta-Correa, Fabré, Berger, SaintPaul, Garcia and Junk2000). It is known for its predatory behavior and is frequently found in association with aquatic vegetation (Petry et al. Reference Petry, Bayley and Markle2003; Sánchez-Botero et al. Reference Sánchez-Botero, Farias, Piedade and Garacez2003). Originally native to the Amazon region (Sousa et al. Reference Sousa, Soares and Prestes2013), S. spilopleura has been introduced into various water bodies across Brazil, including the Tietê, Jacaré-Pepira, and Jacaré-Guaçu Rivers.
Callichthys callichthys (Linnaeus 1758), commonly referred to as tambuatá or tamuatá, belongs to the Callichthyidae family within the order Siluriformes. This species is widely distributed throughout South America, inhabiting freshwater systems from the eastern side of the Andes to as far south as Buenos Aires (Mello et al. Reference Mello, González-Bergonzoni and Loureiro2011). C. callichthys is known as a benthic feeder, displaying significant seasonal variations in habitat use. During winter, it tends to lead a benthic life (Knoppel Reference Knoppel1970; Lowe-McConnell Reference Lowe-McConnell1964), while in summer, it becomes more active and can be found in neritic zones, engaging in nesting behaviors and providing parental care to its offspring (Carter and Beadle Reference Carter and Beadle1931; Lowe-McConnell Reference Lowe-McConnell1964).
Molecular and phylogenetic analysis
Metacercariae were isolated and fixed in ethanol PA (Merck, Darmstadt, Germany), followed by DNA extraction using the DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA, USA) as per the manufacturer’s instructions, resulting in a final volume of 50 μl. Subsequently, DNA samples (5 μl) were subjected to polymerase chain reaction (PCR) amplification using primers specific to the cytochrome c oxidase I (COI) gene barcode region, following the protocol described by Moszczynska et al. (Reference Moszczynska, Locke, McLaughlin, Marcogliese and Crease2009). The PCR reaction mixture (25 μl) contained puReTaq Ready-to-Go Beads (GE Healthcare, Chicago, IL, USA) supplemented with stabilizers (bovine serum albumin and deoxynucleotide triphosphates) and ≈ 2.5 units of puReTaq DNA polymerase.
PCR thermal cycling conditions consisted of an initial denaturation at 94°C for 30 s, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 90 s, and a final extension step at 72°C for 7 min. PCR products were visualized on agarose gel stained with GelRed (Phenix Research, Candler, NC, USA), and bands of interest were purified using the QIAquick PCR Purification Kit (QIAGEN).
Purified DNA was then subjected to direct sequencing using the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on a genetic analyzer (ABI 3500; Applied Biosystems). Sequences were assembled and edited into contigs using Sequencher 5.2.4 (Gene Codes, Ann Arbor, MI, USA), and their identities were verified by comparison using the BLAST program (http://blast.ncbi.nlm.nih.gov).
Sequence alignment was performed using Geneious 7.1.3 (KEARSE et al. 2012), and the best-fit model for nucleotide evolution (GTR+G+I) was determined using the Akaike information criterion in jModelTest (Posada Reference Posada2008). Phylogenetic analyses were conducted using Bayesian Inference (BI) and Maximum Likelihood (ML) methods on the CIPRES Science Gateway (Miller et al. Reference Miller, Schwartz and Pfeiffer2013). Bayesian analysis utilized the settings: lset nst = 6, rates = invariable, ncat = 4, shape = estimate, inferrates = yes, and basefreq = empirical, with MCMC chains run for 10,000,000 generations, sampling one tree every 1,500 generations. The first 25% of generations were discarded as burn-in, and the consensus tree (majority rule) was computed from the remaining trees, considering nodes with posterior probabilities >90% as well supported.
Maximum Likelihood analysis was performed using RAxML (Stamatakis 2014), with bootstrap support values derived from 1,000 replicates, and nodes supported by bootstrap values >70% considered well supported. The resulting phylogenetic trees were visualized using FigTree v.1.3.1 (Rambaut Reference Rambaut2021).
Results
Morphological data
The morphological characteristics of Clinostomum sp. metacercariae from S. spilopleura will be referred as Clinostomum sp. HM41 (Figure 1, Table 1), and the metacercariae found in C. callichthys will be referred as Clinostomum sp. HM125 (Figure 2, Table 1). All measurements are given in micrometers plus the standard deviation.
Figure 1. Clinostomun sp. HM41 metacercarie from Serrasalmus spilopleura (Kner, 1858) collected in the Ibitinga reservoir in the state of São Paulo, Brazil (scale: 2mm).
Table 1. Comparative measurements of Clinostomum sp. HM41 and Clinostomum sp. HM125 and other species reported from fish and also fish-eating birds. Measurements are show in μm with the mean and standard deviation when available
Figure 2. Clinostomun sp. HM125 metacercarie from Callichthys callichthys (Linnaeus, 1758), collected in the Jurumirim reservoir, São Paulo state, Brazil (scale: 2mm).
We collected 62 specimens of Clinostomum sp. HM41 metacercariae (Figure 1) from Serrasalmus spilopleura. These metacercariae were predominantly found infecting various anatomical sites including the musculature, ocular cavity, palate, operculum, and lower jaw. Additionally, we collected 10 specimens of Clinostomum sp. HM125 (Figure 2) from Callichthys callichthys, with the primary site of infection localized in the musculature.
Clinostomum sp. HM41
Taxonomy Summary
Host: Serrasalmus spilopleura specimens collected had mean weight of 209.08 ± 26.42 kg and mean length of 15.2 ± 3.09 cm.
Locality: The Ibitinga reservoir in the state of São Paulo, Brazil (22°30’S; 47°55’W)
Epidemiological data: Prevalence of 80% and mean intensity of 15.5 ± 3.88 parasites per fish, and mean abundance of 12.4 ± 3.02 parasites (range, 4–36 parasites)
Specimens deposit:
Representative DNA: Sequences will be deposited in GenBank after the manuscript acceptance.
Description:
A morphometric description of specimens found in S. spilopleura hosts (n=10) is provided: the oral suction cup exhibited a mean length of 393.64 ± 98.89 and a mean width of 303.07 ± 137.02. Meanwhile, the ventral suction cup had a mean length of 1007.60 ± 235.17 and a mean width of 959.20 ± 185.26. The intestinal cecum measured on average 7287.02 ± 1336.19 in length. The uterus displayed a mean length of 1749.30 ± 377.60 and a mean width of 637.56 ± 260.43. The cirrus sac had a mean length of 314.80 ± 28.56 and a mean width of 299.70 ± 28.07. The ovary measured on average 154.25 ± 48.99 in length and 86.77 ± 39.77 in width. The anterior testis exhibited a mean length of 483.84 ± 161.44 and a mean width of 263.53 ± 71.40, while the posterior testis had a mean length of 473.91 ± 127.79 and a mean width of 363.64 ± 141.31 (Table 1).
Clinostomum sp. HM125
Taxonomy Summary
Host: Callichthys callichthys specimens collected had mean weight of 57.29 ± 12.06 kg and mean length of 12.69 ± 1.01.
Locality: Jurumirim reservoir, São Paulo state (23°12’17’’ S; 49°13’19’’ W)
Epidemiological data: The parasites presented prevalence of 15%, mean intensity of 3.33 ± 1.20 (range, 1–5 parasites).
Specimens deposit:
Representative DNA: Sequences will be deposited in GenBank after the manuscript acceptance.
Description:
A morphometric description of specimens found in C. callichthys hosts (n=3) is provided: the oral suction cup averaged 371.93 ± 68.94 in length and 411.18 ± 112.89 in width, while the ventral suction cup measured 828.09 ± 97.21 in length and 834.31 ± 128.87 in width. The intestinal cecum exhibited an average length of 7020.33 ± 1190.95. The uterus showed dimensions with a mean length of 1233.29 ± 452.53 and a mean width of 220.74 ± 162.63. The cirrus sac had a mean length of 356.12 ± 54.83 and a mean width of 164.85 ± 11.45. The ovary’s average length was 162.65 ± 75.35, and its width was 108.01 ± 70.72. Lastly, the anterior testis measured 345.68 ± 68.02 in length and 325.34 ± 49.80 in width, while the posterior testis had a mean length of 351.47 ± 124.28 and a mean width of 361.76 ± 104.90.
Molecular data
Two mitochondrial COI gene sequences were obtained, measuring 593 base pairs for Clinostomum sp. HM41 (metacercariae) and 595 base pairs for Clinostomum sp. HM125 (metacercariae). Upon performing BLASTn local alignments, Clinostomum sp. HM41 (metacercariae) exhibited 86% similarity to Ithyoclinostomum yamagutii (MN696164) which was the closest related taxa while Clinostomum sp. HM125 (metacercariae) showed 98.7% similarity to Clinostomum sp. Cr_Ha1 (MF673562).
Phylogenetic analyses using Maximum Likelihood and Bayesian methods were conducted on a 412 base pair alignment containing 52 taxa, resulting in similar tree topologies. The phylogenetic tree revealed two main clades labeled as A and B (Figure 3). Clade A included Ithyoclinostomum yamagutii (MN696163-64), along with Ithyoclinostomum species from North and Central America, and Clinostomum sp. HM41 (metacercariae), albeit with low node support (bootstrap 54, posterior probability 0.8) (Figure 3). The genus Euclinostomum showed robust node support, and clade B encompassed a diverse assemblage of Clinostomum species (Figure 3), where Ithyoclinostomum dimorphum (OP174427) clustered within, aligning with previous findings (Simões et al. Reference Simões, Alves, López-Hernández, Couto, Moreira and Pinto2022) regarding its classification as Clinostomum sp.
Figure 3. Maximum Likelihood tree based on barcoding COI sequences of trematodes clinostomids worldwide. GenBank accession numbers are given after species names. Nodal supports values are shown bootstrap (B) and posterior probability (PP), respectively. Values for weakly supported nodes (<0.9 PP and <90 B) are not shown. Gray scale rectangles differentiate main clades and their schematic host figures.
Clinostomum sp. HM125 (metacercariae) clustered closely with species from South America, specifically from Argentina and Brazil, with strong node support (Figure 3). It appears to belong to the same species group as Clinostomum sp. Cr Ha1 (MF673562), Clinostomum sp. Cr Ha2 (MF673563), Clinostomum sp. Adult-Cra (MW187310), Clinostomum sp. Cra1 (MF673556), and Clinostomum sp. Cra1 (MF673557), exhibiting less than 2% intraspecific variation according to BLASTn results. Furthermore, Clinostomum sp. 43 (KJ818259), originally found as metacercariae in guppy (Poecilia reticulata) in Brazil, clustered prominently within the same clade as Clinostomum sp. HM125 and related species from Argentina (Figure 3).
Discussion
Identification of Clinostomum metacercariae solely based on morphological attributes can lead to misidentifications. To enhance the accuracy of Clinostomum systematics and deepen our understanding of the life cycle of these digenetic trematodes, molecular techniques have become indispensable (Bonette et al. Reference Bonett, Steffen, Trujano-Alvarez, Martin, Bursey and McAllister2011; Caffara et al. Reference Caffara, Locke, Gustinelli, Marcogliese and Fioravanti2011; Gustinelli et al. Reference Gustinelli, Caffara, Florio, Otachi, Wathuta and Fioravanti2010; Locke et al. Reference Locke, Caffara, Marcogliese and Fioravanti2015; Matthews and Cribb Reference Matthews and Cribb1998; Sereno-Uribe et al, Reference Sereno-Uribe, Pinacho-Pinacho, García-Varela and Pérez-Ponce de León2013; Simões et al. Reference Simões, Alves, López-Hernández, Couto, Moreira and Pinto2022). Despite the scarcity of morphological studies that differentiate species, molecular DNA sequencing has proven crucial for accurate species differentiation within the genus (Caffara et al. Reference Caffara, Locke, Gustinelli, Marcogliese and Fioravanti2011; Locke et al. Reference Locke, Caffara, Marcogliese and Fioravanti2015; Rosser et al. Reference Rosser, Alberson, Khoo, Woodyard, Pote and Griffin2016).
In 2005, the concept of integrative taxonomy was officially introduced as a comprehensive method for the description of taxa, integrating different data sources and methodologies to reach the final result (Dayrat Reference Dayrat2005; Will et al. Reference Will, Mishler and Wheeler2005). Data on conspecific specimens generated by different researchers can be analyzed comparatively (Patterson et al. Reference Patterson, Cooper, Kirk, Pyle and Remsen2010; Satler et al. Reference Satler, Carstens and Hedin2013; Schlick-Steiner et al. Reference Schlick-Steiner, Seifert, Stauffer, Christian, Crozier and Steiner2007). Within this concept, molecular analysis drives the continuous evolution of taxonomic tools, complementing the fields of morphology, ecology, natural history, and statistics (Knapp Reference Knapp and Wheeler2008). Pante et al. (Reference Pante, Schoelinck and Puillandre2014) argue that integrative taxonomy should be encouraged and developed within the formal description of species, highlighting it as a tool for enhancing and improving the quality of hypotheses concerning species and their descriptions. In Brazil, there are records of at least five species of Clinostomum in larval stage parasitizing fishes (Table 2).
Table 2. Records of species and GenBank entries of the genus Clinostomum and their respective hosts worldwide
However, the vast majority of these records are based on morphological identifications rather than molecular analyses, making it challenging to characterize the distribution of species within the genus in Brazil and South America (Tavares-Dias et al. Reference Tavares-Dias, Silva and Florentino2021). In our work, we are not describing as new species since we do not have the adult phase.
In our morphological analysis, Clinostomum sp. HM41 (metacercariae) exhibited morphological characteristics typical of Clinostomum species rather than Ithyoclinostomum spp., yet molecular analysis grouped it within the Ithyoclinostomum clade (low support), and with more taxa added to the database, this result can possibly change. This discrepancy underscores the need for further sequencing of additional taxa to validate the taxonomy within the genus Ithyoclinostomum. Ithyoclinostomum species, known for their larger body size compared to Clinostomum, were historically distinguished based on morphological features such as cirrus-sac position, testes shape, gonad position, and the free area between the ventral sucker and anterior testis (Rosser et al. Reference Rosser, Woodyard, Mychajlonka, King, Griffin, Gunn and López-Porras2020; Simões et al. Reference Simões, Alves, López-Hernández, Couto, Moreira and Pinto2022). However, recent molecular data challenge these distinctions. For instance, Simões et al. (Reference Simões, Alves, López-Hernández, Couto, Moreira and Pinto2022), evaluating the phylogenetic position of Ithyoclinostomum dimorphum compared to Clinostomum spp. in Brazil, demonstrated that size alone is not a reliable morphological criterion to differentiate Clinostomum spp. from Ithyoclinostomum spp. These observations show that, despite recent advances and applications of molecular techniques in taxonomic studies in South America, trematode species whose descriptions are based solely on morphological characters need to be revisited and analyzed regarding their phylogenetic positions compared to species from other genera, even if morphologically distinct. There are still several issues related to the availability of a molecular database, particularly concerning genera of trematodes, most of whose species have taxonomic descriptions based solely on morphological characters and have not yet been sequenced (Poulin et al. Reference Poulin, Presswell and Jorge2020; Simões et al. Reference Simões, Alves, López-Hernández, Couto, Moreira and Pinto2022).
Clinostomum sp. HM125 (metacercariae), found in Callichthys callichthys from Jurumirim reservoir, clustered together with Argentinean species, suggesting conspecificity with minor intraspecific variation. Phylogenetic analyses highlight a distinct clustering pattern of these haplotypes across South America. Figure 3 illustrates how South American Clinostomum species within clade B1 are grouped, also incorporating species from Central and North America. Presently, 23 species are validated within the genus Clinostomum, with 11 found in the New World. These species exhibit distribution patterns across South America: C. marginatum (Rudoplhi 1819), C. detrucatum (Braun 1899), C. heluans (Braun 1899), and C. fergalliarii (Montes, Barneche, Pagano, Ferrari, Martorelli, and Pérez-Ponce de León 2021); Central America: C. heluans (Braun 1899), C. tataxumui (Sereno-Uribe, Pinacho-Pinacho, García-Varela & Pérez-Ponce de León 2013), C.arquus (Sereno-Uribe, García-Varela, Pinacho-Pinacho & Pérez-Ponce de León 2018), C. caffarae (Sereno-Uribe, García-Varela, Pinacho-Pinacho and Pérez-Ponce de León 2018), and C. cichlidorum (Sereno-Uribe, García-Varela, Pinacho-Pinacho & Pérez-Ponce de León 2018); and North America: C. attenuatum (Cort 1913), C. album (Rosser Alberson, Woodyard, Cunningham, Pote and Griffin 2017), C. poteae (Rosser, Baumgartner, Alberson, Noto, Woodyard, King, Wise and Griffin 2018), and C. marginatum (Montes, Barneche, Pagano, Ferrari, Martorelli, and Pérez-Ponce de León 2021).
The expanding occurrence of Clinostomum parasites in new localities suggests an increase in their distribution range, likely facilitated by migratory piscivorous birds (Antonucci et al. Reference Antonucci, Souza, Ramos, Casasli and Ribeiro2015). This study underscores ongoing taxonomic challenges in trematode diagnostics and emphasizes the pivotal role of molecular tools in encompassing these issues. Furthermore, it broadens the understanding of Clinostomum spp. metacercariae distribution in South America while raising pertinent questions regarding the validity of the genus Ithyoclinostomum.
In conclusion, our study underscores the complexity of Clinostomum taxonomy and the necessity for integrating molecular tools alongside traditional morphological approaches to accurately identify and differentiate species within this genus. The unexpected molecular clustering of Clinostomum sp. HM41 (metacercariae) within the Ithyoclinostomum clade can change in the future studies, while more taxa will be added to the database, also highlighting unresolved taxonomic issues regarding Ithyoclinostomum characterization and calling for further investigations involving a broader range of taxa. The phylogenetic insights gained from our study, particularly regarding Clinostomum sp. HM125 (metacercariae) and its clustering with Argentinean species, contribute to understanding regional species diversity, distribution, and evolutionary dynamics within Clinostomum. Moving forward, continued collaborative efforts across disciplines will be essential to elucidate the intricate relationships among Clinostomum species, refine species boundaries, and deepen our understanding of their ecological roles and impacts on host communities worldwide.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X24000993.
Availability of data and material
All data supporting the findings of this study are available within the paper.
Authors contribution
Conceptualization: FFJ, RKA, VDA. Methodology: FFJ, MIM, FHY, VDA. Formal analysis: MIM, FHY. Investigation: FFJ, LARL, VDA. Writing – Original Draft: TG, FFJ, LARL, VDA. Writing – Review & Editing: LARL, MIM, FHY, RJS, RKA, VDA. Supervision: VDA.
Funding
The authors thank the São Paulo Research Foundation (Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP) for financial support (process nº 2016/21040-9); the Coordination of Superior Level Staff Improvement (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES) M.I.M. grant number AUX-PE-PNPD 3005/2010; The Young Researcher Program (Programa Jovem Pesquisador – PROPE-UNESP 02/2016; and the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq) F.H.Y. processes nº 304502/2022-7 and 174814/2023-2).
Consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interest
The authors declare no competing interests.
Ethical standards
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