Is Saccocoelioides bacilliformis Szidat, 1973 (Digenea: Haploporidae) a valid species? Genetic and morphological studies of this controversial species

Abstract

Saccocoelioides is a genus within the family Haploporidae and is distributed throughout the Americas. The recent application of molecular techniques has facilitated the reorganization of this genus and the description of new species, resulting in a current total of 28 species. In Argentina, 11 species have been identified; however, the validity of Saccocoelioides bacilliformis and Saccocoelioides octavus remains controversial. Recently, specimens of digeneans were collected from two distinct geographical locations: the Paraná River basin (Misiones province) and the Río de la Plata basin (Buenos Aires province). These specimens were found parasitizing two different fish species: Bryconamericus sylvicola and Bryconamericus iheringi. Sequences of the 28S and COI genes were obtained for both digeneans from the two collection sites and hosts. The morphological study, combined with genetic sequences, confirmed their assignment to the genus Saccocoelioides. The COI and 28S gene sequences obtained from specimens at both collection sites exhibited genetic distances of 0.13% and 3.41%, respectively, indicating that these Saccocoelioides specimens represent a single species. The comprehensive taxonomic study of the newly collected specimens, along with an examination of the type material of S. bacilliformis and S. octavus, allows us to confirm the validity of both species. Argentina is home to more than 550 species of freshwater fauna, and it is likely that new species of Saccocoelioides remain to be discovered in underexplored regions of the basin, such as the Uruguay, Pilcomayo and Bermejo rivers.

Introduction

Saccocoelioides is a genus within the Haploporidae family distributed throughout the Americas. According to Curran et al. (2018), Saccocoelioides species can be divided into two groups or morphotypes, the diminutive and the robust, based on body size, the number and ratio of mature eggs size to pharynx size, and the extent of the hindbody occupied by the uterus. However, Montes et al. (2024) reexamined these characters, discarding body size as a distinguishing feature, and found that uterine extension and egg number were ambiguous. Consequently, they redefined both groups based on a single character: the relative size of mature eggs (with occulated miracidia) to the length of the pharynx. Thus, the ‘diminutive group’, consisting of 20 species, is characterized by larger eggs, while the ‘robust group’, with small eggs, includes 8 species.

Ten species of Saccocoelioides have been documented in Argentina, including two newly described species: Saccocoelioides kirchneri Martorelli, Montes, Barneche, Reig Cardarella & Curran, 2022 and Saccocoelioides miguelmontesi Montes, Croci, Barneche, Ferrari, Reig Cardarella & Martorelli, 2024. Five of these species belong to the ‘diminutive group’: Saccocoelioides carolae Lunaschi, 1984, S. kirchneri, S. magnivatus Szidat, 1954, S. nanii Szidat, 1954 and S. octavus Szidat, 1970. The remaining five species belong to the ‘robust group’, consisting of Saccocoelioides antonioi Lunaschi, 1984, S. elongatus Szidat, 1954, S. magnus Szidat, 1954, S. miguelmontesi and S. szidati Travassos et al., 1969 (Curran et al. 2018; Ostrowski de Nuñez et al., 2017; Curran et al., 2018; Martorelli et al., 2022; Montes et al., 2024).

There is a controversy regarding two species, Saccocoelioides bacilliformis Szidat, 1973 and S. octavus. Lunaschi (2002), after morphologically studying the types of both species, concluded that they are synonyms, with the latter species, S. octavus, being the valid because of priority, whereas S. bacilliformis remains a junior synonym. In their review of haploporid species, Curran et al. (2018) agreed with Lunaschi´s (2002) conclusion. However, Gallas & Utz (2019) revalidated S. bacilliformis by examining material collected from Astyanax aff. fasciatus (Cuvier) and Astyanax henseli Melo & Buckup from Pinada Island, Rio Grande do Sul, Brazil. A significant limitation of Szidat’s (1970, 1973) descriptions is the inability to ascertain the identity of the host species. Szidat identified the host of S. octavus as Astyanax fasciatus (Cuvier), and the host of S. bacilliformis as Astyanax bipunctatus (Linnaeus). In a recent review of Astyanax spp., Terán et al. (2020) recognized A. fasciatus as Psalidodon fasciatus Cuvier. However, according to Mirande and Koerber (2020), this species is not present in Argentina. Furthermore, neither author mentions ‘A. bipunctatus’ in their studies. When consulted ichthyologists Serra Alanis and Terán (personal communications) suggested that the ‘Astyanax fasciatus’ mentioned by Szidat (1970) could be Psalidodon rutilus (Jenyns), whereas the ‘A. bipunctatus’ represents an unknown species. It is possible that Szidat (1973) misinterpreted the name of the species (‘bipunctatus’ meaning ‘two spots’) and that it could in fact be Astyanax bimaculatus (Linnaeus) (‘bimaculatus’ also meaning ‘two spots’). If this is the case, the species most similar in general appearance and present in the region is likely to be Astyanax lacustris (Lütken) or Astyanax abramis (Jenyns), according to Serra Alanis (personal communication).

As part of the studies conducted in the provinces of Buenos Aires and Misiones, specimens resembling S. bacilliformis in morphology were found. Therefore, the objective of this work was to study the new specimens collected through an integrative taxonomic approach, including the reporting of the 28S and COI genes. Additionally, the type material of S. octavus and S. bacilliformis was examined to facilitate comparison between the specimens and to determine their taxonomic status.

Material and Methods

Collection of samples and morphological study

Using hand nets, specimens of Bryconamericus silvicola Braga were collected in the Mbocay stream (Paraná River basin, Misiones province), and Bryconamericus iheringi (Boulenger) in the Juan Blanco stream (La Plata River basin, Buenos Aires province) (Fig. 1). They were transported alive to the field laboratory or to CEPAVE in plastic bags containing water from the sampling site, supplemented with oxygen. Upon arrival, the fishes were euthanized by cervical dissection, necropsied, and examined for parasites. Digeneans were found in the intestine of the fish, and subsequently removed, placed in Petri dishes, heat-killed with hot water and fixed in 10% formalin for morphological studies. Some specimens were preserved in cold 96% ethanol for later DNA extraction.

Figure 1. Site of collection of Saccocoelioides spp in Argentina. Legends: 1. Saccocoelioides antonioi (Boca Cerrada, Punta Lara), 2. Saccocoelioides bacilliformis (Reconquista river), 2*. Saccocoelioides bacilliformis (ex Bryconamericus iheringi, Juan Blanco stream), this study, 2**. Saccocoelioides bacilliformis (ex Bryconamericus sylvicola, Mbocay stream, Misiones province), this study, 3. Saccocoelioides carolae (from a- Los Talas; b-Chascomús lagoon, no one defined as type locality), 4. Saccocoelioides elongatus (La Plata River basin, in Buenos Aires City), 4´. Saccocoelioides elongatus (Middle Paraná River basin, Corrientes province), 5. Saccocoelioides kirchneri (El Bosque, La Plata), 6. Saccocoelioides magniovatus (Lujan River), 7. Saccocoelioides magnus (Rosario, Middle Paraná River basin), 8. Saccocoelioides miguelmontesi (Iguazú River), 9. Saccocoelioides nanii (ex Prochilodus lineatus, Middle Paraná River basin), 9´. Saccocoelioides nanii (ex Hypostomus commersoni, Talavera Island, Irigoyen channel, Middle Paraná River basin), 9´´. Saccocoelioides nanii (ex Prochilodus lineatus, Saladita lagoon, La Plata River basin), 9´´´. Saccocoelioides nanii (ex Prochilodus lineatus, Mar Chiquita, Cordoba province), 10. Saccocoelioides octavus (Chascomús lagoon), 11. Saccocoelioides szidati (ex Schizodon fasciatus and Megaleporinus obtusidens Middle Paraná River, in front of Rosario City, Santa Fe province).

Morphological analysis

Whole-mount specimens were processed following standard parasitological techniques and stained with hydrochloric carmine (Pritchard & Kruse, 1982). Measurements and digital images of the specimens were captured using an Olympus Bx51 microscope equipped with an AmScope MU 1000 10 MP digital camera. The structures were measured using ImageJ software (Schneider et al., 2012). Drawings were created using the photos obtained from the stained specimens with the help of the Photopea program (https://www.photopea.com). Measurements are presented as the mean followed by minimum and maximum values in parentheses and are given in micrometers (μm). Voucher specimens were deposited in the Helminthological Collection of the Museo de La Plata (MLP-He), La Plata and in the Parasitological Collection of the Museo Argentino de Ciencias Naturales ‘Bernardino Rivadavia’ (MACN-Pa), Buenos Aires, Argentina. Additionally, the type specimens of S. bacilliformis (MACN-Pa 59/1-5) and S. octavus (MACN-Pa 112/1-3), deposited in the Parasitological Collection of the Museo Argentino de Ciencias Naturales ‘Bernardino Rivadavia’, Buenos Aires, Argentina, was studied for comparison.

DNA extraction, amplification and sequencing

Total genomic DNA was extracted from Saccocoelioides sp. using PURO-Genomic DNA kit (PB-L Productos Bio-Logicos S.A., Argentina) following the manufacturer’s protocol. Amplification of each gene was conducted using polymerase chain reaction (PCR) on an Eppendorf Mastercycler thermal cycler (Hamburg, Germany). The fragment of the 28S rDNA gene (28S) was amplified with the forward primer LSU-5 (5′-TAG GTC GAC CCG CTG AAY TTA AGC A-3′) and the reverse primer 1500R (5′-GCT ATC CTG AGG GAA ACT TCG-3′) as described by Tkach et al. (2003). PCR thermocycling conditions followed Tkach et al. (2003). The fragment of the mtDNA COI gene (COI) was amplified using the primer pair DICE 1F (5′-ATT AAC CCT CAC TAA ATT WCN TTR GAT CAT AAG-3′) and DICE 14R (5′-TAA TAC GAC TCA CTA TAC CHA CMR TAA ACA TAT GAT G-3′) as described by Van Steenkiste et al. (2015). PCR conditions were following Montes et al. (2024). Both strands of each PCR product were sequenced using an ABI 3730XLs sequencer (Macrogen Inc.). The sequences were assembled using Geneious v.6.0.5 (http://www.geneious.com) and utilized to search for homologous sequences in GenBank (Table 1).

Table 1. Collection data and GenBank accession numbers for Saccocoelioides spp. analyzed in this study; new sequences in bold

Sequence comparison and phylogenetic analysis

Sequences were aligned using the online version of MAFFT v.7 (Katoh et al., 2019). The best partitioning scheme and substitution model for the DNA partition were chosen based on the Bayesian Information Criterion (Schwarz, 1978) using the ’greedy’ search strategy in Partition Finder v.1.1.1 (Lanfear et al., 2014). The optimal nucleotide substitution models were HKY+G for the 28S sequences, and F81+I, GTR+G, and TrN+G for the first, second, and third codon positions of the COI sequences, respectively.

Phylogenetic reconstruction was conducted using Bayesian Inference (BI) in MrBayes v.3.2.3 (Ronquist et al., 2012). Phylogenetic trees were generated using two parallel analyses of Metropolis-Coupled Markov Chain Monte Carlo for 20 million generations each to estimate the posterior probability (PP) distribution. Topologies were sampled every 1,000 generations, and the average standard deviation of split frequencies was maintained below 0.01, as recommended by Ronquist et al. (2012). The robustness of the clades was evaluated using Bayesian PP, with PP > 0.95 considered strongly supported. A majority consensus tree with branch lengths was constructed for each run after discarding the initial 25% sampled trees.

The proportion (p) of absolute nucleotide sites (p-distance) (Nei & Kumar, 2000) was calculated to compare the genetic distance between lineages. The p-value matrix was obtained using MEGA X (Kumar et al., 2018), employing the bootstrap method (1000 replicates) and uniform rate nucleotide substitution (transition + transversions). Newly generated sequences (Table 1) were submitted to the National Center for Biotechnology Information (NCBI) GenBank database (http://www.ncbi.nlm).

Results

Morphological analysis

Saccocoelioides bacilliformis Szidat, 1973

(Fig. 2, Table 2, Supplementary material 1-3)

Table 2. Measurements of Saccocoelioides octavus and Saccocoelioides bacilliformis according different authors (measurements of the new specimens in bold)

Abbreviations: B, body; E, egg; Ex, external; H, hermaphroditic; In, internal; L, length; O, oral; Oes, oesophagus; Ov, ovary; P, pharynx; Ppx, prepharynx; S, sucker; Sem, seminal; T, testicle; V, ventral; Ves, vesicle; W, width.

Figure 2. Saccocoelioides bacilliformis collected from pyloric caecum of Bryconamericus iheringi. Scale bar = 200 μm.

Description

Measurements based on type material (three gravid specimens) and eight new specimens, three from B. iheringi (La Plata Basin) and five from B. sylvicola (Parana Basin). Body elongate, entirely spinous, with pigment present in anterior half of body, wide at ventral sucker level. Oral sucker subterminal. Ventral sucker in the anterior third of the body. Prepharynx as long as the pharynx. Oesophagus 3 times longer than the pharynx. Ceca sac-shaped, terminating blindly in pre-testicular zone. Testis rounded, in the middle of the body. Hermaphroditic sac oval. External seminal vesicle globose. Internal seminal vesicle longer than wide. Genital pore opens between ventral sucker and pharynx. Ovary slightly elongated longitudinally. Laurer’s canal and Mehlis’ gland not observed. Uterus extending between hermaphroditic sac and posterior body end. Vitellarium arranged in two clusters of follicles extending from posterior level of ventral sucker to anterior margin of testis. Fully developed eggs containing miracidia with eye spots. Excretory vesicle Y-shaped (not drawn).

Taxonomic summary

Type host: Astyanax bimaculatus (L.) (probably Astyanax lacustris (Lütken) or Astyanas abramis (Jenyns)).

Other hosts: Bryconamericus silvicola Braga and Bryconamericus iheringi (Boulenger).

Site of infection: pyloric caecum.

Type locality: Río Reconquista, La Plata River basin (Buenos Aires province).

Other localities: Mbocay stream, Paraná River basin (Misiones province) and Juan Blanco stream, La Plata River basin (Buenos Aires province).

Prevalence of infection: 94% (17/18) in B. sylvicola; 4%, (9/37) in B. iheringi.

Mean intensity and abundance: 10.18 and 9.61 in B. sylvicola; 2.22 and 0.54 in B. iheringi, respectively.

Type material: holotype MACN-Pa 59/1 (27918a); paratypes MACN-Pa 59/2 (27918b), MACN-Pa 59/3 (27918c), MACN-Pa 59/4 (27918d), MACN-Pa 59/5 (27918e).

Additional material deposited: MLP-He 8139 (four gravid specimens) and MACN-Pa 804/1-3 (six gravid specimens) from B. sylvicola; MLP-He 8140 (three gravid specimens) and MACN-Pa 803 (one gravid specimen) from B. iheringi.

Saccocoelioides octavus Szidat, 1970

(Table 2, Supplementary material 4)

Description

Measurements based on type material (two gravid specimens). Body elongate, entirely spinous, with pigment in anterior half of body, wider below ventral sucker. Ventral sucker in the anterior third of the body. Prepharynx half the length of the pharynx. Oesophagus two times longer than the pharynx. Ceca sac-shaped, not reaching pre-pretesticular region. Testis rounded, in the middle of the body. Hermaphroditic sac oval, external seminal vesicle and internal seminal vesicle not measured. Genital pore opens between ventral sucker and pharynx. Ovary slightly elongated longitudinally. Laurer’s canal and Mehlis’ gland not observed. Uterus extending between hermaphroditic sac and posterior end of testis. Vitellarium arranged in two clusters of follicles extending from posterior level of ventral sucker to anterior margin of testis. Eggs fully developed containing miracidia with eye spots. Excretory vesicle Y-shaped.

Taxonomic summary

Type host: Psalidodon rutilus (Jenyns) (syn = Astyanax fasciatus (L.).

Site of infection: Pyloric caecum.

Type locality: Chascomús lagoon, Buenos Aires Province.

Type material: MACN-Pa 112/1 (27973a), MACN-Pa 112/2 (27973b), MACN-Pa 112/3 (27973c).

Remarks

The objective of this manuscript is to critically evaluate the synonymy proposed by Lunaschi (2002) between S. bacilliformis and S. octavus. This evaluation is based on a comprehensive study of the type material of both species, as well as newly collected specimens of S. bacilliformis. Furthermore, for both species under investigation, we present measurements and size relationships that are currently crucial for species discrimination. These include the percentage of body width to body length, the oral sucker to ventral sucker ratio, the oral sucker to pharyngeal ratio, the post-caecal and post-testicular space, and others (see Table 2).

The measurements obtained by Lunaschi (2002) of the type material of S. octavus and S. bacilliformis are in general agreement with those previously reported by Szidat (1970, 1973). However, these descriptions are brief and incomplete. The type material of S. octavus comprises 10 specimens, of which only two are gravid, whereas in the case of S. bacilliformis, the type material is composed of 12 specimens, of which only three have eggs (see Supplementary material 1-5). The measurements obtained in this study are within the range provided by Szidat (1970, 1973) and Lunaschi (2002). The slight differences that can be observed can be attributed to the inclusion of juvenile specimens in the measurements of both authors. Moreover, the specimens deposited by Szidat (1970,1973), particularly those of S. octavus, have been overstained, making it challenging to observe certain structures, such as the hermaphrodite sac or the seminal vesicle, with the desired clarity.

In a recent publication, Gallas & Urtz (2019) proposed to revalidate S. bacilliformis based on a morphological study of specimens found parasitizing Astianax aff. fasciatus (Cuvier) and A. henseli Melo & Buckup in Brazil. The specimens exhibit an elongated body of consistent width and appear to be slightly smaller, yet still within the range of measurements presented by the type material described by Szidat (1973). The authors posit that the observed differences between the S. bacilliformis specimens may be attributed to the greater intraspecific variation than previously observed or to the fixation method (Gallas & Urtz, 2019).

The new specimens studied, which were found parasitizing B. silvicola and B. iheringi, exhibit similarities to one another and to the type material of S. bacilliformis (see Supplementary Material 1, 2, 3, and 5). The new specimens appear to be slightly larger; however, the observed meristic differences could be attributed to fixation methods or the low number of B. iheringi specimens measured (n=3). Nevertheless, these morphological differences are minimal, as evidenced by the size ratios between structures in the new specimens and the type material (see Table 2), and therefore do not justify the creation of a new species.

Finally, from the study of the type material of both species and the newly collected specimens of S. bacilliformis, morphological differences between S. octavus and S. bacilliformis can be observed. These differences include body morphology (pyriform vs. elongated, respectively), the relative percentage of body width to body length (33% vs. 16-27%, respectively), the ratio of the oral sucker to the ventral sucker width (1:0.73 for S. bacilliformis and 1:1.32 in S. octavus), egg length (106 μm vs. 82 μm, respectively), and the presence of eyespots (absent vs. present, respectively), among other characteristics.

Molecular results

Partial sequences of the 28S gene from S. bacilliformis were obtained from one specimen on B. iheringi (1358 bp) and one from B. sylvicola (1357 bp). These new sequences, combined with those obtained from GenBank (Table 1), were used to construct a matrix with 102 terminals and 1337 bp. In the 28S tree reconstructed through Bayesian analysis (Fig. 3), S. bacilliformis clustered within the ‘robust species’ group (sensu Curran et al. 2018) and formed the sister branch of the clade containing S. magnus, S. elongatus and S. miguelmontesi. The ‘robust group’ clade is strongly supported, with posterior probabilities exceeding 0.80. The genetic distance and number of nucleotide differences among the partial sequences of Saccocoelioides spp. ranged from 0 to 5.75% and from 0 to 44 bp, respectively. The two populations of S. bacilliformis from B. iheringi and B. sylvicola showed a p-distance of 0.13%. Saccocoelioides bacilliformis, S. elongatus, S. magnus, and S. miguelmontesi were separated by a distance of 1.32-4.30% and 10-29 bp of nucleotide difference (Table 3).

Figure 3. Phylogram resulting from Bayesian Inference (20,000,000 generations) of partial 28S rDNA gene sequences of Saccocoelioides bacilliformis rooted by Forticulcita sp. Branch support values indicate posterior probabilities ≥80%. Grey bar (A) = group A (ex ‘diminutive group’ according to Curran et al., 2018), Black bar (B) = group B (ex-robust form from Curran et al., 2018).

Table 3. Uncorrected p-distances of the 28S rDNA gene expressed as percentage among Saccocoelioides spp. calculated in MEGA X are below the diagonal line. The number of nucleotide difference is above the diagonal line. New sequences reported in this study are in bold

Abbreviations: Bi = Bryconamericus iheringi, Bs = Bryconamericus sylvicola, ID = intragroup p-distance, n/c = not calculated.

The relations observed in the 28S phylogenetic tree among the Saccocoelioides spp. are similar to those reported by Montes et al. (2024). The most significant differences are seen in the relative positions of Saccocoelioides beauforti (Hunter & Thomas, 1961) Overstreet, 1971, Saccocoelioides chauhani Lamothe- Argumedo, 1976, Saccocoelioides olmecae Andrade-Gómez, Pinacho-Pinacho, Hernández-Orts, Sereno-Uribe & García-Varela, 2016 and Saccocoelioides sogandaresi Lumsden, 1963 However, these differences are found in clusters with low posterior probability, despite the fact that the cluster including all of them together has a posterior probability of 96%.

Partial sequences of COI from S. bacilliformis were obtained, comprising two from B. iheringi (564-600 bp) collected in Juan Blanco stream (La Plata River basin, Buenos Aires province) and three from B. sylvicola (573-606 bp) sampled in Mbocay stream (Paraná River basin, Misiones province). These new sequences, combined with those obtained from GenBank (Table 1), resulted in a matrix consisting of 52 terminals and 623 bp.

In the COI tree (Fig. 4), S. bacilliforis from B. iheringi and B. sylvicola share a clade with high support, and both are the sister species of S. miguelmontesi in the ‘roboust group’. The p-distances and number of nucleotide differences among Saccocoelioides spp. varied from 10.84% to 23.27% and from 32 to 75 bp, respectively. The sequences obtained from each sample site were identical to each other’s (Table 4, ID column). The p-distance between S. bacilliformis populations is 3.41% with 11 bp of difference, whereas compared with S. miguelmontesi, it shows a p-distance of 19.5% to 20.12% and 63 to 65 bp of difference (Table 4).

Figure 4. Phylogram resulting from Bayesian Inference (20,000,000 generations) of COI mtDNA gene sequences of Saccocoelioides bacilliformis rooted by Forticulcita platana. Branch support values indicate posterior probabilities ≥80%. Grey bar (A) = group A (ex “diminutive form” from Curran et al., 2018), Black bar (B) = group B (ex “robust form” according to Curran et al., 2018).

Table 4. Uncorrected p-distances of the COI mtDNA gene expressed as percentage calculated in MEGA X using variance estimation, the Bootstrap Method (500 replicates), and uniform nucleotide substitution rates (transition + transversion) below the diagonal line

The number of nucleotide difference is shown above the diagonal line.

Abbreviations: Bi = Bryconamericus iheringi, Bs = Bryconamericus sylvicola, ID = intragroup p-distance.

In the COI tree obtained in the present study, S. olmecae clusters with Saccocoelioides orosiensis Curran, Pulis, Andres, & Overstreet, 2018, S. chauhani and Saccocoelioides macrospinosus Andrade-Gómez, Sereno-Uribe, & García-Varela, 2019. However, in Montes et al., (2024), this species is closely related to Saccocoelioides cichlidorum (Aguirre-Macedo & Scholz, 2005) Garcia Varela, Andrade-Gómez, & Pinacho-Pinacho, 2017, and Saccocoelioides tkachi Curran, Pulis, Andres, & Overstreet, 2018. In the present tree, S. macrospinosus is related to S. chauhani and S. orosiensis, but in Montes et al., (2024), it is positioned at the base of the clade formed by S. chauhani, S. orosiensis, S. olmecae, S. cichlidorum and S. tkachi. Additionally, the posterior probabilities of the clades differ between the studies. In Montes et al., (2024), the well-supported clades include S. olmecae, S. cichlidorum and S. tkachi, while in the recent tree, S. olmecae clusters with other species with low posterior probability. Another difference is the position of S. macrospinosus, which has a posterior probability 84% in the new tree, clustering with S. orosiensis and C. chauhani in this study. However, as mentioned earlier, the position of this species in Montes et al., (2024) is more basal, relative to other species of the “diminutive group” except Saccocoelioides lamothei Aguirre-Macedo & Violante-González, 2008.

Discussion

The species S. bacilliformis and S. octavus were considered valid until a study by Lunaschi (2002) synonymized them without evaluating the morphological differences between the specimens of both species. Subsequently, differing views emerged following the establishment of the synonymy. On the one hand, Kohn et al., (2007) acknowledged the synonymy proposed by Lunaschi (2002) while continuing to recognize both species. Conversely, Curran et al., (2018), who studied the type material — though only that of S. octavus (see Figs. 21-23) — upheld the validity of the synonymy but emphasized the need for molecular analysis. Later, Gallas & Utz (2019) revalidated S. bacilliformis using new specimens collected from Astyanax spp. in southern Brazil. However, these authors did not study the type material nor did they obtain genetic information for the species.

The comparative morphological study of S. octavus and S. bacilliformis supports the validity of both species, despite the lack of genetic information on S. octavus. Curran et al., (2018) defined two groups or morphotypes (‘diminutive’ and ‘robust’) within Saccocoeliodes. In this context, S. octavus could belong to the ‘diminutive group’ (small body, few large eggs and extension of the uterus in the hindbody). On the other hand, S. bacilliformis should belong to the ‘diminutive group’ based solely on the size of the eggs relative to the size of the pharynx. However, the larger body size (greater than >1.7 mm), the uterus being predominantly in the anterior body, and the large number of eggs place the species in the ‘robust group’.

Furthermore, S. bacilliformis is genetically related to the ‘robust group’. Recently, Montes et al., (2024) found that S. miguelmontesi also belongs genetically to the ‘robust group’, despite having eggs of similar length to the pharynx, a small body size, and a uterus mainly in the anterior body (reaching the posterior edge of the testis). Thus, according to the results obtained, the groups defined by Curran et al., (2018) appear to be artificial and need to be revised, as discrepancies arise when comparing morphological and genetic characteristics. To simplify this classification, it is proposed to label the ’small body’ group as Group A and the ’robust body’ as group B. These groups are defined mainly on the basis of genetic analysis of the COI and 28S genes (see Figs. 3, 4).

Using the number of eggs (many small eggs or few large eggs) to define group membership is also ambiguous, as the maturity of the digenean determines the number of eggs. Similarly, the extension of the uterus has been used as a character to distinguish between groups. For example, the ‘diminutive group’ shows restriction to the posterior body, whereas the ‘robust group’ shows extension into the anterior body or remains completely within the posterior body. These results show that the separation of the ‘diminutive group’ and the ‘robust group’ based on morphology does not align with the separation of the species into ‘Group A’ and ‘Group B’ based on genetic results, highlighting the need to find additional morphological characters for effective discrimination between species groups.

A significant challenge for both Saccocoelioides species has been determining the identity of the type host. As previously outlined, none of the species identified as type hosts by Szidat (1970, 1973) are currently present in Argentina. However, in the case of S. octavus, this issue appears to have been resolved, as the type host referenced by Szidat is not A. fasciatus (not native to Argentina), but rather P. rutilus (a species indigenous to the country). The case of S. bacilliformis is distinct, with the identity of the type host remaining a point of contention. This is likely because of a typographical error, as the species referenced by Szidat (1973), ‘Astyanax bipunctatus’, is not recognized under that name. Additionally, some authors have proposed that it may have been Astyanax bimaculatus, a species not native to Argentina. Given the similarities, it seems probable that the host in question was A. abramis or A. lacustris. However, confirmation of this hypothesis requires the discovery of S. bacilliformis in these hosts.

It is noteworthy that S. bacilliformis exhibits a broad geographic distribution and a wide host spectrum, including at least five species of fish. The type locality of the Reconquista River (type host) and the Juan Blanco stream (B. iheringi) are situated within the La Plata River basin, whereas the Mbocay stream (B. sylvicola) is located 1,600 km from the type locality, within the Parana River basin. However, both the La Plata River and the Parana River are part of the Parano-Platense basin, one of the largest in South America. In contrast, the specimens documented by Gallas & Urtz (2019) in Astyanax aff. fasciatus and A. henseli belong to a different basin (Guaiba River basin) in Brazil. However, genetic data is required to confirm whether these specimens can be identified as S. bacilliformis. The prevalence of infection also appears to be higher in B. sylvicola (94%) than in the other hosts, suggesting that it could be a primary host, followed by A. henseli (40%), A. aff. fasciatus (8.7%) and B. iheringi (4%). The intensity of infection was very high for Astyanax aff. fasciatus (1-54 individuals) and B. sylvicola (1-30 individuals) (see Gallas & Urtz, 2019 and present study). The presence of several hosts is common in some Saccocoelioides species, such as S. cichlidorum (Aguirre-Macedo & Scholz, 2005) Andrade-Gómez, Pinacho-Pinacho, & García-Varela, 2017, S. lamothei Aguirre-Macedo & Violante-González, 2008, S. miguelmontesi Montes, Croci, Barneche, Ferrari, Reig Cardarella, & Martorelli, 2024, S. orosiensis Curran, Pulis, Andres, & Overstreet, 2018 and S. sogandaresi Lumsden, 1963 (Curran et al., 2018; Andrade-Gómez et al., 2017; Montes et al., 2024). Szidat’s (1970) note on the species specificity of the genus and high host specificity seems invalid in the case of S. bacilliformis.

The distribution of Saccocoelioides spp. reported so far extends from the Salado River (province of Buenos Aires) northward through the Paraná River basin. Surprisingly, there are no records of Saccocoelioides spp. in the Uruguay River or in northwestern Argentina (Bermejo and Pilcomayo Rivers), although these rivers, together with the Paraná and La Plata Rivers, form the Parano-Platense basin. Several hypotheses could explain this absence. One possibility is that Saccocoelioides spp. are actually absent from these basins, but this seems unlikely given the low number of host fish species studied, especially considering that more than 550 freshwater fish species have been recorded in Argentina (Mirande & Koerber, 2020). It is hoped that a greater sampling effort will lead to the discovery of new species of this genus, especially in the Uruguay, Pilcomayo and Bermejo rivers.

In conclusion, our study confirms the validity of S. bacilliformis and S. octavus. However, S. octavus requires further study based on the collection of new specimens for morphological and molecular analyses. Genetic information plays a crucial, sometimes indispensable role in clarifying the true affiliation of digeneans. Finally, the number of Saccocoelioides species reported is now 28, including 12 in Argentina.