Introduction
The Iguazu National Park was established as a biological reserve in 1934 to protect the natural and cultural resources of the Paranaense subtropical forest (Administración de Parques Nacionales, 2020). This area includes the Iguazú River Basin, which is divided by the Iguazu waterfalls into two ecoregions: the Iguazu and the Lower Parana ecoregions, located upstream and downstream of the waterfalls, respectively (Abell et al., Reference Abell, Thieme and Revenga2008). These ecoregions show differences in fish community composition and a high diversity of endemic fish species (Casciotta et al., Reference Casciotta, Almirón and Ciotek2016).
During a parasitological survey of fishes conducted in the Arrechea Stream, which drains into the upstream region of the Iguazú River (Iguazu ecoregion), allocreadiid trematodes were collected from the intestines of the characiform fish Characidium heirmostigmata Graça & Pavanelli, 2008. The family Allocreadiidae Looss, 1902 is distributed worldwide and their members are mainly parasites of the digestive tract of freshwater fishes (see Caira & Bogéa, Reference Caira, Bogéa, Jones, Bray and Gibson2004). The studied specimens belong to a group of nine genera of allocreadiids characterized by having muscular lobes associated with the oral sucker, including Acrolichanus Ward, 1917; Auriculostoma Scholz, Aguirre-Macedo & Choudhury, 2004; Crepidostomum Braun, 1900; Creptotrema Travassos, Artigas & Pereira, 1928; Creptotrematina Yamaguti, 1954; Bunodera Railliet, 1896; Bunoderella Schell, 1964; Megalogonia Surber, 1928; and Pseudoparacreptotrema Pérez Ponce de León, Pinacho-Pinacho, Mendoza-Garfias, Choudhury & García-Varela, 2016 (Atopkin et al., Reference Atopkin, Sokolov, Vainutis, Voropaeva, Shedko and Choudhury2020; Pérez-Ponce de León et al., Reference Pérez-Ponce de León, Sereno-Uribe, García-Varela, Mendoza-Garfias, Hernández-Mena, Pinacho-Pinacho and Choudhury2020). However, our specimens conform with the diagnosis of the genus Auriculostoma in that they possess a single pair of oral lobes, funnel-shaped oral sucker and pretesticular uterus.
The genus Auriculostoma currently contains nine species distributed in Neotropical freshwater fishes across the Americas. Two species are found in Mexico: Auriculostoma lobata Hernández-Mena, Lynggaard, Mendoz-Garfias & Pérez-Ponce de León, 2016 in Brycon guatemalensis Regan (Characiformes: Bryconidae); and Auriculostoma totonacapanensis Razo-Mendivil, Mendoza-Garfias, Pérez-Ponce de León & Rubio-Godoy, 2014 in Astyanax mexicanus (de Filippi) (Characiformes: Characidae) (Razo-Mendivil et al., Reference Razo-Mendivil, Mendoza-Garfias, Pérez-Ponce de León and Rubio Godoy2014; Hernández-Mena et al., Reference Hernández-Mena, Lynggaard, Mendoza-Garfias and Pérez-Ponce de León2016). Two species are found in Central America: Auriculostoma astyanace Scholz, Aguirre-Macedo & Choudhury, 2004 in Astyanax fasciatus Cuvier (= Astyanax aeneus) (Characiformes: Characidae) from Nicaragua; and Auriculostoma tica Hernández-Mena, Pinacho-Pinacho, García-Varela, Mendoza-Garfias & Pérez- Ponce de León, 2018 in Gymnotus maculosus (Albert & Miller) (Gymnotiformes: Gymnotidae) from Costa Rica (Scholz et al., Reference Scholz, Aguirre-Macedo and Choudhury2004; Hernández-Mena et al., Reference Hernández-Mena, Pinacho-Pinacho, García-Varela, Mendoza-Garfias and Pérez-Ponce de León2019). The remaining five species occur in South America: Auriculostoma diagonale Curran, Tkach & Overstreet, 2011 in Stethaprion cf. erythrops Cope (Characiformes: Characidae) from Peru; Auriculostoma foliaceum Curran, Tkach & Overstreet, 2011 in Bryconops cf. caudomaculatus (Günther) (Characiformes: Characidae) from Peru; Auriculostoma stenopteri (Mañé-Garzón & Gascón, 1973) Scholz, Aguirre-Macedo & Choudhury, 2004 in Charax stenopterus Cope (Characiformes: Characidae) from Uruguay; Auriculostoma platense (Szidat, 1954) Scholz, Aguirre-Macedo & Choudhury, 2004 in Pimelodus maculatus Lacépède (= Pimelodus clarias) from Brazil; and Auriculostoma macrorchis (Szidat, 1954) Scholz, Aguirre-Macedo & Choudhury, 2004 in Pachyurus bonariensis Steindachner (Perciformes: Sciaenidae) from Argentina (Mañé-Garzón & Gascón, Reference Mañé-Garzón and Gascon1973; Kohn et al., Reference Kohn, Fernandes and Cohen2007; Curran et al., Reference Curran, Tkach and Overstreet2011; Ostrowski de Nuñez et al., Reference Ostrowski de Nuñez, Arredondo and Gil de Pertierra2017).
In Argentina, two species of Auriculostoma have been reported: A. macrorchis and A. platense. The former species has been mainly found in siluriforms, particularly in Ageneiosus inermis (L.), Ageneiosus militaris Valenciennes, Auchenipterus nigripinnis (Boulenger), Auchenipterus osteomystax (Miranda Ribeiro) (=Auchenipterus nuchalis (Spix & Agassiz)) (Siluriformes: Auchenipteridae); also in Luciopimelodus pati (Valenciennes) (Siluriformes: Pimelodidae), Rhinodoras dorbignyi (Kner) (Siluriformes: Doradidae) from La Plata River, Buenos Aires City; Middle Paraná River, Corrientes Province; Colastiné River (tributary of the Paraná River), Santa Fe Province; and Paraná-Guazú River, Entre Ríos Province. Auriculostoma platense has been reported also from siluriforms, in Iheringichthys labrosus (Lütken), Pimelodus albicans (Valenciennes), Pimelodus argenteus Perugia, P. maculatus Lacépède (= P. clarias) (Siluriformes: Pimelodidae) and R. dorbignyi (Siluriformes: Doradidae); in Rhamphichthys rostratus (Linnaeus) (Gymnotiformes: Rhamphichthyidae) from La Plata River, Buenos Aires City and Colastiné River, Santa Fe Province (Szidat, Reference Szidat1954; Ostrowski de Nuñez et al., Reference Ostrowski de Nuñez, Arredondo and Gil de Pertierra2017). The newly sampled specimens of Auriculostoma are different from the previously described congeners. The main objective of this study is to describe a new species of Auriculostoma from characiforms of the Iguazu National Park by using molecular and morphological evidence.
Materials and methods
Sample collections and morphological study
The Arrechea Stream runs near the Macuco Path, ending in a 23-m waterfall in the Iguazu National Park, Misiones Province, Argentina, Iguazu Ecoregion. Specimens of C. heirmostigmata were sampled from this stream in 2018 and 2019. Fish were collected with hand nets and transported alive to the field laboratory where they were necropsied. Digeneans found in the intestine were heat-killed and preserved in a 10% formalin. Whole-mount specimens were stained with hydrochloric carmine and mounted in Canada balsam according to standard parasitological techniques (Pritchard & Kruse, Reference Pritchard and Kruse1982) and one specimen was used for scanning electron microscope (SEM) studies. Digital images of specimens were taken using an AmScope MU 1000 10 MP digital camera (Irvine, USA)attached to the microscope and structures were measured using the ImageJ software (Schneider et al., Reference Schneider, Rasband and Eliceiri2012). Drawings were made using a light microscope Olympus BX53 (Tokyo, Japan) fitted with differential interference contrast and equipped with a camera lucida. Measurements are given in micrometres (μm) as the mean ± standard deviation followed by the minimum and maximum in parenthesis.
Some specimens were preserved in 96% molecular grade ethanol for DNA analysis. In addition, a single specimen morphologically identified as Auriculostoma cf. stenopteri found in C. stenopterus from Punta Lara, Buenos Aires Province, Argentina, was preserved in 96% ethanol and subsequently used for DNA analysis.
Molecular analysis
Partial sequences of 28S rDNA (28S rRNA gene) were obtained through the polymerase chain reaction (PCR), using the forward primer LSU-5 (5′-TAG GTC GAC CCG CTG AAY TTA AGC A-3′) (Littlewood et al., Reference Littlewood, Curini-Galletti and Herniou2000) and reverse primer 1500R (5′-GCT ATC CTG AGG GAA ACT TCG-3′) (Tkach et al., Reference Tkach, Littlewood, Olson, Kinsella and Swiderski2003). The PCR reactions were carried out in an Eppendorf Mastercycler thermal using Green GoTaq 5X Buffer (Promega, Madison, Winsconsin, USA), 2.5 mm magnesium chloride (Promega), 0.2 mm of New England Biolabs (Ipswich, Massachusetts, USA) Nucleotide Mix and Flexi GoTaq polymerase enzyme (Promega). The thermocycling profile followed that of Tkach et al. (Reference Tkach, Littlewood, Olson, Kinsella and Swiderski2003).
Phylogenetic analysis
Sequences obtained in this study were assembled using the platform Geneious Pro version 5.1.7 (Drummond et al., Reference Drummond, Ashton, Buxton, Cheung, Cooper, Duran and Wilson2016), and used to search for homologous sequences in GenBank (table 1) using a BLAST search. Sequences (table 1) were aligned using the online version of MAFFT version 7 (Katoh & Standley, Reference Katoh and Standley2013) and a matrix of 1246 bp constructed. The online program Gblocks version 0.91 (Castresana, Reference Castresana2000; Talavera & Castresana, Reference Talavera and Castresana2007) using a less stringent selection (allowing smaller final blocks, gap position within the final blocks and less strict flanking positions) was applied to detect ambiguously aligned, hypervariable regions in the 28S rDNA dataset, according to a secondary structure model. The best partitioning scheme and substitution model for the DNA partition were chosen under the Bayesian Information Criterion (Schwarz, Reference Schwarz1978) using the ‘greedy’ search strategy in Partition Finder version 1.1.1 (Lanfear et al., Reference Lanfear, Calcott, Ho and Guindon2012, Reference Lanfear, Calcott, Kainer, Mayer and Stamatakis2014). The obtained sequences were aligned with previously published sequences of Auriculostoma spp. in the GenBank database along to species of other three genera of allocreadiids, and the callodistomid Prosthenhystera obesa (Diesing, 1850) Travassos, 1922 as outgroup for rooting the trees (table 1); the appropriate nucleotide substitution model implemented for the 28S rDNA matrix was TVM + I+G.
Table 1. Collection data and GenBank accession numbers for allocreadiid species analysed in this study. New species in bold.
The phylogenetic reconstruction was carried out using Bayesian inference through MrBayes version 3.2.3 (Ronquist et al., Reference Ronquist, Teslenkovan and van der Mark2012). Phylogenetic trees were constructed 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 1000 generations and the average standard deviation of split frequencies was observed to be less than 0.01 (Ronquist et al., Reference Ronquist, Teslenkovan and van der Mark2012). The robustness of the clades was assessed using Bayesian PP, where PP > 0.95 was considered strongly supported. A majority consensus tree with branch lengths was reconstructed for each run after discarding the first 25% of sampled trees.
Additionally, the uncorrected p-distance (Nei & Kumar, Reference Nei and Kumar2000) was obtained to compare the genetic distance between lineages. The P-value matrix was obtained using MEGA X (Kumar et al., Reference Kumar, Stecher, Li, Knyaz and Tamura2018), with the bootstrap method (1000 replicates). Newly generated sequences were submitted to the National Centre for Biotechnological Information (NCBI) GenBank database (https://www.ncbi.nlm.nih.gov), under the GenBank accession numbers MN822004–MN822005.
Results
Allocreadiidae (Looss, 1902)
Auriculostoma Scholz, Aguirre-Macedo & Choudhury, 2004
Auriculostoma guacurarii n. sp.
Taxonomic summary
Host. Characidium heirmostigmata (Graça & Pavanelli) (Characiformes: Crenuchidae).
Locality. Arrechea Stream, Iguazu River above the waterfalls, Iguazu National Park, Misiones, Argentina (25°39′29″S, –54°27′15″W).
Site of infection. Intestine.
Prevalence, intensity and abundance. 40% (12/30), 1.08, 0.44.
Type material. Museo de Ciencias Naturales de La Plata, Helminthology Collection: holotype (MLP-He 7713) and paratypes (MLP He 7714).
ZooBank registration. urn:lsid:zoobank.org:pub:C4F63F63-0EA4-4711-B8FC-C078F43134B5.
Etymology. The species name guacurarii is given in honour of Andresito Guacurari, a forgotten hero from the Indigenous Guarani People, who bravely fought against the Portuguese invasion of Argentina in the 19th century. It also honours the jaguar (Panthera onca Linnaeus, 1758) Guacurarí, which was killed by poachers in the Urugua-í Reserve, Misiones Province, Argentina. We aim to raise awareness about the importance of forests in Misiones as biodiversity hotspots and the need for their protection.
Description
Based on six adult specimens (fig. 1a). Body elongate, 1254 ± 147 (1084–1439) long, 372 ± 83 (259–456) wide; maximum width at the ventral sucker level; tegument smooth, eyespot remnants present. Forebody 30–32% of body length. Oral sucker subterminal, with one pair of muscular oral lobes on both sides, 112 ± 19 (93–137) long, 129 ± 23 (106–160) wide, bearing small dome-like papillae (arrangement not clearly seen (fig. 2a, b). Ventral sucker spherical, muscular, slightly larger than oral sucker, 138 ± 15 (122–157) long, 132 ± 14 (120–148) wide. Oral sucker/ventral sucker length and width ratio 1:1.15–1.23, 1:0.92–1.06, respectively. Prepharynx absent. Pharynx muscular, globular 51 ± 9 (41–60) long, 58 ± 10 (50–69) wide. Oesophagus straight, 62 ± 19 (41–76) long. Intestinal bifurcation in forebody, half distance between ventral and oral suckers. Caeca long, ending between posterior testis and posterior end of body. Post-caecal space length 219 ± 25 (200–256). Testes two, oblique, smooth, post-equatorial, intercaecal. Anterior testis dextral, 134 ± 6 (128–139) long, 71 ± 3 (68–74) wide. Posterior testis larger than anterior testis, 152 ± 22 (126–173) long, 74 ± 8 (69–83) wide. Post-testicular space length 340 ± 56 (270–401), representing 27 (25–30) per cent of body length. Cirrus sac 241 ± 79 (158–316) long, dorsal, medial, slightly curved, extending from the genital pore to the anterior margin of ovary, containing elongated and bi-partite seminal vesicle, with constriction between pars prostatica and seminal vesicle (fig. 1b). Cirrus not observed. Genital pore opening medially on forebody, close to intestinal bifurcation. Ovary oval, equatorial, sinistral, pretesticular, 125 ± 22 (101–145) long, 88 ± 17 (77–104) wide. Seminal receptacle post-ovarian, subspherical, 54 ± 9 (47–63) long, 66 ± 24 (45–92) wide. Mehlis’ gland and Laurer's canal not observed. Vitelline follicles extending from level of intestinal bifurcation to almost reach posterior end of body, filling intra and extracaecal space. Uterus pre- and inter-testicular, extending from mid-level of testes to the genital atrium; eggs few in number, 67 ± 2 (65–70) long, 40 ± 1 (38–42) wide. Excretory vesicle I-shaped, 127 ± 17 (103–143).
Fig. 1. (a) Auriculostoma guacurarii n. sp., ventral view of holotype; (b) detail of the genital terminalia in dorsal view.
Fig. 2. Detail of Auriculostoma guacurarii n. sp. oral sucker. (a) SEM photo. (b) Optical microscope photo. White asterisk = papillae only observable in that photo. Black asterisk = papilla observed on both photos. L, muscular lobes.
Taxonomic remarks
The new species agrees with the diagnosis of Auriculostoma by possessing a single pair of muscular lobes on either side of the oral sucker (Scholz et al., Reference Scholz, Aguirre-Macedo and Choudhury2004; Razo Mendivil et al., Reference Razo-Mendivil, Mendoza-Garfias, Pérez-Ponce de León and Rubio Godoy2014; Hernández-Mena et al., Reference Hernández-Mena, Pinacho-Pinacho, García-Varela, Mendoza-Garfias and Pérez-Ponce de León2019). Unfortunately, the specimen used for SEM was in poor shape and the structure of the oral lobes is not clearly seen. The new species is part of a group of six species of Auriculostoma that possess testes with smooth margins, including A. astyanace, A. diagonale, A. platense, A. tica and A. totonacapanensis (Scholz et al., Reference Scholz, Aguirre-Macedo and Choudhury2004; Curran et al., Reference Curran, Tkach and Overstreet2011; Razo Mendivil et al., Reference Razo-Mendivil, Mendoza-Garfias, Pérez-Ponce de León and Rubio Godoy2014; Hernández-Mena et al., Reference Hernández-Mena, Pinacho-Pinacho, García-Varela, Mendoza-Garfias and Pérez-Ponce de León2019). Auriculostoma astyanace differs from A. guacurarii n. sp. by having testes in tandem, a genital pore located between the anterior margin of the ventral sucker and the caecal bifurcation, and by parasitizing fishes of the family Characidae in Costa Rica and Nicaragua (Scholz et al., Reference Scholz, Aguirre-Macedo and Choudhury2004). The remaining four species possess oblique testes as in the new species.
Auriculostoma guacurarii n. sp. can be distinguished from A. diagonale, a species described from characids in Peru, by having larger eggs (65–70 vs. 55 μm) vitelline follicles extending anteriorly to the level of the caecal bifurcation instead of reaching anteriorly to the pharynx level, and by having a cirrus sac extending posteriorly to the level of ovary, and not to the anterior testis level. The new species differs from A. platense, a species occurring in siluriform fishes of Brazil and Argentina, in the extension of the vitelline follicles (caecal bifurcation level vs. mid-level of the oesophagus), genital pore position near or over the caecal bifurcation vs. between the anterior margin of the ventral sucker and caecal bifurcation, egg size (65–70 vs. 75 μm) and extension of cirrus sac (reaching to the level of the ovary vs. not reaching the posterior margin of the ventral sucker). In addition, in Argentina A. guacurarii n. sp. is found in characiforms of the family Crenuchidae in the Iguazu River, whereas A. platense occurs in Siluriformes of the Parana River. Auriculostoma guacurarii n. sp. differs from A. tica, a species recently described from a gymnotid freshwater fish in Costa Rica, in the length of body (1084–1439 vs. 1551–2118), in the position of the genital pore, which opens at the level of the caecal bifurcation rather than between the pharynx and the caecal bifurcation, and in the extension of the caeca (half-distance between posterior testis and posterior end of body vs. almost reaching the posterior end of body). Finally, A. guacurarii n. sp. closely resembles A. totonacapanensis in the size of most structures, in having smooth and oblique testes, in the position of the genital pore (at the level of the caecal bifurcation), in having a cirrus sac originating in the ovarian region, in having vitelline follicles not confluent in the post-testicular area and in the fact that both are parasites of characiforms, although A. totonacapanensis is found in the species of characid with the most northern distribution range, the Mexican tetra, A. mexicanus De Filippi, in central Mexico. However, in the new species, the oral and ventral suckers are smaller, the post-testicular space is larger, the eggs are larger and the intestinal caeca extend beyond the posterior testis (in A. totonacapanensis intestinal caeca reach the level of the posterior margin of posterior testis).
Two species of Auriculostoma have been reported from freshwater fishes in Argentina – A. macrorchis and A. platense. The new species differs from A. macrorchis by having oblique testes rather than testes in tandem, by the anterior extension of the vitelline follicles that reach the level of the caecal bifurcation rather than to pharynx level, and by host association because A. macrorchis is mainly a parasite of siluriforms whereas the new species is a parasite of characiforms. The differences between the new species and A. platense are mentioned above. We report the presence of A. stenopteri in Argentina for the first time. We obtained a single specimen of this species from the intestine of C. stenopterus; the specimen was identified morphologically and the whole specimens was used for DNA. We decided to refer the species as Auriculostoma cf. stenopteri provisionally until more specimens are collected from the same host and locality, and vouchers can be deposited in a parasite collection.
Molecular characterization
The results of the morphological study were further corroborated by sequencing the 28S rRNA gene. Only the reverse sequence and one forward and reverse sequence pair of the partial fragment of 28S rDNA gene were recovered for A. guacurarii n. sp. (1139 bp) and A. cf. stenopteri (1178 bp), respectively. The obtained phylogram provides strong support for the monophyly of the genus Auriculostoma (fig. 3). Two main clades are formed within Auriculostoma, one containing A. lobata and A. astyanace, a highly supported group (PP 100%), and other clade containing A. totonacapanensis as the sister species of A. cf. stenopteri and A. guacurarii n. sp. + A. tica, the clade is supported with a high PP value (99%). The genetic divergence of the 28S rDNA gene between the new species and the other congeners for which sequences are available varied between 2.11 and 3.46% (table 2). The results of the molecular analysis further corroborated the independence of the new species.
Fig. 3. Phylogram resulting from Bayesian inference of partial 28S rDNA gene sequences of Auriculostoma guacurarii n. sp. and Auriculostoma cf. stenopteri among allocreadiids rooted with Prosthenhystera obesa. Scale bar shows the number of subtitutions per site.
Table 2. Genetic divergence among species of allocreadiids relevant to the study, estimated through of uncorrected p-distance of the 28S rDNA gene and expressed as percentage. Intraspecific divergence in bold.
OG, outgroup; n/c, not calculated.
The topology of the phylogenetic tree, regarding on the position of Auriculostoma, Creptotrematina and Wallinia, agrees with previous studies by Curran et al. (Reference Curran, Tkach and Overstreet2011), Razo-Mendivil et al. (Reference Razo-Mendivil, Mendoza-Garfias, Pérez-Ponce de León and Rubio Godoy2014) and Alves Dias et al. (Reference Alves Dias, Pérez-Ponce de León, de Almeida Camargo, Müller, da Silva, Kozlowiski de Azevedo and Abdallah2020), but disagrees with Pérez-Ponce de León et al. (Reference Pérez-Ponce de León, Razo-Mendivil, Mendoza-Garfias, Rubio-Godoy and Choudhury2015), Hernández-Mena et al. (Reference Hernández-Mena, Lynggaard, Mendoza-Garfias and Pérez-Ponce de León2016, Reference Hernández-Mena, Pinacho-Pinacho, García-Varela, Mendoza-Garfias and Pérez-Ponce de León2019) and Da Silva et al. (Reference Da Silva, Dias, da Silva and Yamada2021). There is no doubt about the monophyly of each genus, but the phylogenetic relationships between them are not well supported and require further scrutiny.
Discussion
The combined morphological features along with the genetic differences exhibited by the specimens of Auriculostoma collected from C. heirmostigmata allow us to validate them as a new species. Auriculostoma guacurarii n. sp. is the tenth species described for this genus, the sixth record of this genus from South America and the first one from the Iguazu Basin. Moreover, it is the first species of Auriculostoma found parasitizing the family Crenuchidae.
Although the sequenced specimen of A. cf. stenopteri was immature, it possessed a single pair of muscular oral lobes, and testes in tandem with irregular margins. Interestingly, it was collected from the type-host species (C. stenopterus) and type locality (La Plata River) where the species was originally described (Mañé-Garzón & Gascón, Reference Mañé-Garzón and Gascon1973; Ostrowski de Nuñez et al., Reference Ostrowski de Nuñez, Arredondo and Gil de Pertierra2017). This was important in deciding that this specimen was conspecific with A. stenopteri, even though we observed that both ventral and oral suckers were almost identical in size; although, in the original description, A. stenopteri was diagnosed as having a ventral sucker larger than the oral sucker. Nevertheless, we only sampled one individual that was also genetically distinct from the new species we describe in this study. The phylogenetic tree and genetic divergence values unequivocally show that the new species represent an independent lineage. Additionally, the tree suggests that testes shape (smooth or lobed) and position (oblique or in tandem), irrespective of their diagnostic value, are characters that do not define natural groupings since they are found independently within the two well-defined monophyletic clades. Nevertheless, we are aware that DNA sequences are only available for six out of the ten species of the genus. The full understanding of the evolutionary and biogeographical history of this Neotropical genus of trematode will benefit from obtaining DNA sequence data of the remaining four species – that is, P. macrorchis, P. platense (both from Argentina), P. diagonale and P. foliaceum (both from Peru). The genus seems to have evolved along with two major groups of Neotropical freshwater fishes, the characiforms and the siluriforms, especially the former, since seven of the ten species are parasites of characiforms, two of siluriforms and one of gymnotids (see Kohn et al., Reference Kohn, Fernandes and Cohen2007; Choudhury et al., Reference Choudhury, Aguirre-Macedo, Curran, Ostrowski de Nuñez, Overstreet, Pérez-Ponce de León and Portes Santos2016; Ostrowski de Nuñez et al., Reference Ostrowski de Nuñez, Arredondo and Gil de Pertierra2017). Previously, Razo-Mendivil et al. (Reference Razo-Mendivil, Mendoza-Garfias, Pérez-Ponce de León and Rubio Godoy2014) proposed that Auriculostoma, along with two other genera of allocreadiids, Wallinia and Creptotrematina, formed a group associated with characiforms. We expect more species of these three genera to be described from South America as more species of characiforms and their parasites are investigated in this region using integrative taxonomy.
Acknowledgements
We are grateful to the Administración de Parques Nacionales, Argentina, for granting us the permit for fieldwork at Iguazu National Park; the Facultad de Ciencias Naturales y Museo (UNLP) and Centro de Estudios Parasitológicos y de Vectores (CEPAVE) for providing us with the laboratory facilities; Jorge Casciotta, Liliana Ciotek, Pablo Giorgis and the staff of CIES and Delegación Regional Noreste for field assistance; and Patricia Sarmiento and Maria Marcia Montes for the SEM photos and line drawings, respectively.
Financial support
This work was partially funded by Consejo Nacional de Investigación Cienífica y Tecnológica (CONICET) (S.R.M., PIP-0015), Fondo para la Investigación Científica y Tecnológica (FONCyT) (M.M.M., BID PICT 2016-4153) and CEPAVE (PUE 3334/16), and also partially funded by the Consejo Nacional de Ciencia Y Tecnología, Mexico (G.P.P.L., CONACyT number A1-S-21694).
Conflicts of interest
None.
Ethical approval
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.
