Introduction
The diversity of amphibians in Brazil is extraordinarily high (1,222 species) (Segalla et al. Reference Segalla, Berneck, Canedo, Caramaschi, Cruz, Garcia, Grant, Haddad, Lourenço, Mângia, Mott, Nascimento, Toledo, Werneck and Langone2021; Frost Reference Frost2023). Most of this diversity comprises anurans with 1,178 species, followed by caecilians (39 species) and salamanders (five species) (Segalla et al. Reference Segalla, Berneck, Canedo, Caramaschi, Cruz, Garcia, Grant, Haddad, Lourenço, Mângia, Mott, Nascimento, Toledo, Werneck and Langone2021; Frost Reference Frost2023). Despite this high biodiversity, the diversity of amphibian parasites is underestimated (Campião et al. Reference Campião, Morais, Dias, Aguiar, Toledo, Tavares and Silva2014; Alcantara et al. Reference Alcantara, Ferreira-Silva, Forti, Morais and Silva2021, Reference Alcantara, Ebert, Müller, Úngari, Ferreira-Silva, Emmerich, Santos, O’Dwyer and Silva2022). The genus Crossodactylus Duméril and Bibron, Reference Duméril and Bibron1841 presently includes 13 species of frogs distributed across South America (Frost Reference Frost2023). One of these species, Schmidt’s Spinythumb frog, Crossodactylus schmidti Gallardo, Reference Gallardo1961 (Anura: Hylodidae Günther, 1858), was first described in Argentina (Misiones) by Gallardo (Reference Gallardo1961) and later reported in Paraguay (southeastern Itapúa province) and Brazil (western Paraná, western Santa Catarina, and northern and western Rio Grande do Sul) (Frost Reference Frost2023). Crossodactylus schmidti is currently considered a federally threatened species, catalogued as Near Threatened (NT) in the IUCN Red List (IUCN 2023), as their populations are declining since the anuran occurs in severely fragmented environments. Data on parasites associated with this anuran species include only one study carried out by Forti et al. (Reference Forti, Pontes, Alcantara, Morais, Silva, Dodonov and Toledo2020), in which the presence of helminths was reported as a part of an ecological study aimed at reporting the infection by chytrid fungus in relation to forest cover, although macroparasites were not taxonomically identified to species level. One of them was morphologically identified as belonging to the trematode genus Creptotrema Travassos, Artigas & Pereira, Reference Travassos, Artigas and Pereira1928.
The genus Creptotrema includes parasites of freshwater teleosts and anurans distributed across the Neotropical region (Franceschini et al. Reference Franceschini, Aguiar, Zago, Yamada, Ebert and Silva2021; Liquin et al. Reference Liquin, Gilardoni, Cremonte, Saravia, Cristóbal and Davies2022). To date, the genus includes 20 valid species (Franceschini et al. Reference Franceschini, Aguiar, Zago, Yamada, Ebert and Silva2021; Liquin et al. Reference Liquin, Gilardoni, Cremonte, Saravia, Cristóbal and Davies2022), with 16 of those occurring in South America – that is, the type-species Creptotrema creptotrema Travassos, Artigas & Pereira, Reference Travassos, Artigas and Pereira1928, Creptotrema conconae Franceschini, Aguiar, Zago, Yamada, Ebert & Silva, Reference Franceschini, Aguiar, Zago, Yamada, Ebert and Silva2021, Creptotrema diagonale (Curran, Tkach & Overstreet, Reference Curran, Tkach and Overstreet2011), Creptotrema foliaceum (Curran, Tkach & Overstreet, Reference Curran, Tkach and Overstreet2011), Creptotrema guacurarii Montes, Barneche, Croci, Balcazar, Almirón, Martorelli & Pérez-Ponce de León, Reference Montes, Barneche, Croci, Balcazar, Almirón, Martorelli and Pérez-Ponce de León2021, Creptotrema lamothei Curran, Reference Curran2008, Creptotrema lynchi Brooks, Reference Brooks1976, Creptotrema macrorchis (Szidat, Reference Szidat1954), Creptotrema megacetabulare Franceschini, Aguiar, Zago, Yamada, Ebert & Silva, Reference Franceschini, Aguiar, Zago, Yamada, Ebert and Silva2021, Creptotrema ocloye (Liquin, Gilardoni, Cremonte, Saravia, Cristóbal & Davies, Reference Liquin, Gilardoni, Cremonte, Saravia, Cristóbal and Davies2022) n. comb., Creptotrema pati Lunaschi, Reference Lunaschi1985, Creptotrema paraense Vicente, Santos & Souza, Reference Vicente, Santos and Souza1978, Creptotrema platense (Szidat, Reference Szidat1954), Creptotrema schubarti Franceschini, Aguiar, Zago, Yamada, Ebert & Silva, Reference Franceschini, Aguiar, Zago, Yamada, Ebert and Silva2021, Creptotrema stenopteri (Mañé-Garzón & Gascón, Reference Mañé-Garzón and Gascón1973), and Creptotrema sucumbiosa Curran, Reference Curran2008. Creptotrema lynchi is the only species reported from an anuran host, Rhinella marina (Linnaeus, 1758) (= Bufo marinus), in Colombia (Brooks, Reference Brooks1976), although the definitive hosts of Creptotrema spp. are preferentially freshwater fishes belonging to multiple orders (Characiformes, Gymnotiformes, Perciformes, and Siluriformes). A new species of Auriculostoma Scholz, Aguirre-Macedo & Choudhury, Reference Scholz, Aguirre-Macedo and Choudhury2004 was described from Argentina in 2022 as a parasite of siluriforms, named A. ocloya (Liquin et al. Reference Liquin, Gilardoni, Cremonte, Saravia, Cristóbal and Davies2022). Apparently, the authors were unaware of the publication by Franceschini et al. (Reference Franceschini, Aguiar, Zago, Yamada, Ebert and Silva2021) in which the genus Auriculostoma was synonymized with Creptotrema. Our study offers a possibility to test for the phylogenetic position of that species within Allocreadiidae Looss, 1902.
Although molecular tools and bioinformatics have revolutionized biodiversity research and can be used to address some of the Linnaean shortfalls (Rubio-Godoy and Pérez-Ponce de León Reference Rubio-Godoy and de León G2023; Vergara-Asenjo et al. Reference Vergara-Asenjo, Fermín, Alfaro and Pizarro-Araya2023), parasite fauna is still considered largely unknown, especially in tropical regions (Dobson et al. Reference Dobson, Lafferty, Kuris, Hechinger and Jetz2008; Carlson et al. Reference Carlson, Dallas, Alexander, Phelan and Phillips2020a). This taxonomic gap holds back the progress of ecological knowledge on parasites and impairs conservation actions (Carlson et al. Reference Carlson, Hopkins, Bell, Doña, Godfrey, Kwak, Lafferty, Moir, Speer, Strona, Torchin and Wood2020b). Even though parasite taxonomy is in crisis with only small groups of prolific authors describing new species (Poulin and Presswell Reference Poulin and Presswell2022), the recent discovery of new species of Creptotrema and other parasite groups is due to the utilization of integrative approaches and the use of distinct tools for morphological analyses, such as light and scanning electron microscopy, and molecular investigations, with the employment of different markers, and phylogenetic analyses.
As a part of a survey on the helminth parasite fauna of Brazilian anurans, we had an opportunity to study the internal organs of C. schmidti in Paraná, Rio Grande do Sul and Santa Catarina states; among them, some individuals were identified as belonging to the genus Creptotrema. A further detailed morphological assessment of the specimens, accompanied by molecular analyses (28S rDNA and COI mtDNA genes) confirmed they represented an undescribed species of trematode parasitizing the near-threatened frog C. schmidti. The new species is described herein, and its phylogenetic position within the genus Creptotrema is tested. Our results expand the knowledge of the interaction of Creptotrema species with amphibian hosts in South America.
Materials and methods
Host sampling and parasitological procedures
Twenty-seven specimens of C. schmidti were sampled in three national parks of Brazil: the Parque Nacional do Iguaçu, municipality of Céu Azul (25°9’4.036"S, 53°50’28.777"W); Parque Estadual Rio Guarani, municipality of Três Barras do Paraná, Paraná state (25°26’42.871"S, 53°9’37.879"W); and Parque Estadual do Turvo, municipalities of Derrubadas (27°8’31.68"S, 53°52’39.35"W) and Frederico Westphalen (27°21’43.36"S, 53°24’38.32"O), Rio Grande do Sul state, and municipality of São Miguel do Oeste (26°45’36.10"S, 53°31’30.47"O), Santa Catarina state. The frogs were euthanized with 2% lidocaine hydrochloride. Frogs were dissected, and all internal organs were screened for parasites under a stereomicroscope.
Trematodes were collected from the small intestine of the anurans, and two fresh specimens were transferred directly to 99.8% ethanol for molecular study while the other specimens were fixed in alcohol-formalin-acetic acid solution under light pressure of a coverslip for 10 min and transferred to 70% alcohol for further processing. At the laboratory, trematodes were stained with alcoholic chloride carmine solution, cleared with eugenol, and analyzed in a computerized system for image analysis (V3 Leica Application Suite, Leica Microsystems, Wetzlar, Germany) in a microscope with differential interference contrast. Morphological descriptions followed the recommendations of Travassos et al. (Reference Travassos, Artigas and Pereira1928) and Fernandes and Kohn (Reference Fernandes and Kohn2014), and the observations provided by Scholz et al. (Reference Scholz, Aguirre-Macedo and Choudhury2004) and Razo-Mendivil et al. (Reference Razo-Mendivil, Pérez-Ponce de León and Rubio-Godoy2014b). Measurements of the specimens are presented as the values of the holotype followed by the range in parentheses (reported in micrometers). Illustrations of the structures were produced with the aid of a camera lucida mounted on a Leica DMLS microscope with phase-contrast optics.
Holotype and paratypes of the new species of Creptotrema were deposited in the Helminthological Collection of the Oswaldo Cruz Institute (CHIOC – Holotype: number 40421a; Paratypes: numbers 40421b, 40422, 40423, 40424, 40425), Rio de Janeiro State, Brazil. The host specimens (C. schmidti) were deposited at the Museu de Zoologia ‘Prof. Adão José Cardoso’ of the Universidade Estadual de Campinas (Unicamp), Campinas, São Paulo, Brazil (ZUEC24284 to 24295).
DNA extraction, amplification, and sequencing
Genomic DNA was extracted from two Creptotrema specimens using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, California, United States), following the manufacturer’s protocol. Fragments of the 28S rDNA gene and the COI mtDNA gene were amplified using the primers and cycling conditions described in Franceschini et al. (Reference Franceschini, Aguiar, Zago, Yamada, Ebert and Silva2021). Conventional polymerase chain reaction (PCR) amplifications were performed on a final volume of 25 μl containing 12,5 μl of 2× MyFiTM Mix (Bioline, Taunton, MA, USA), 3.0 μl of extracted DNA, 7.5 μl of pure water, and 1.0 μl of each PCR primer. PCR products (2.0 μl) were run on an agarose gel (1%) using GelRedTM fluorescent nucleic acid dye and loading buffer to confirm amplicon size and yield. PCR amplicons were purified using the QIAquick PCR Purification Kit (Qiagen), following the manufacturer’s instructions. Automated sequencing was performed directly on purified PCR products using a BigDye v.3.1 Terminator Cycle Sequencing Ready Reaction kit on an ABI 3500 DNA genetic sequencer (Applied Biosystems). Forward and reverse sequences were assembled and edited using Sequencher v. 5.2.4 (Gene Codes, Ann Arbor, MI, USA).
Phylogenetic analyses
To perform the phylogenetic analyses, two independent datasets were created: the first contained the newly generated 28S rDNA sequences, published sequences of Allocreadiidae retrieved from GenBank, and sequences of Prosthenhystera Travassos, 1922 (Callodistomidae Odhner, 1910), Dicrocoelium Dujardin, 1845 (Dicrocoeliidae Looss, 1899), Degeneria Campbell, 1977 (Gorgoderidae Looss, 1899), and Phyllodistomum Braun, 1899 (Gorgoderidae), which were used as outgroups (Table S1). The second dataset contained the newly generated COI mtDNA sequence, published sequences of Allocreadiidae retrieved from GenBank, and sequences of Phyllodistomum parasiluri Yamaguti, 1934 (Gorgoderidae), Dicrocoelium dendriticum (Rudolphi, 1819), and Dicrocoelium chinensis (Sudarikov & Ryjikov, 1951) Tang & Tang, 1978 (Dicrocoeliidae) as outgroups (Table S1).
The alignments of the two datasets were performed separately using the MUSCLE algorithm implemented on Geneious 7.1.3 (Kearse et al. Reference Kearse, Moir, Wilson, Stones-Havas, Cheung, Sturrock, Buxton, Cooper, Markowitz, Duran, Thierer, Ashton, Meintjes and Drummond2012) with default settings. The presence of stop codons and indels for the COI mtDNA alignment was verified by amino acid translation using the trematode mitochondrial code table on Geneious 7.1.3 (Kearse et al. Reference Kearse, Moir, Wilson, Stones-Havas, Cheung, Sturrock, Buxton, Cooper, Markowitz, Duran, Thierer, Ashton, Meintjes and Drummond2012). Prior to the phylogenetic analyses, the best-fitting model of nucleotide substitution for the aligned datasets was selected in the JModelTest software (Posada Reference Posada2008) using the Akaike information criterion, as GTR + G + I for the 28S rDNA dataset and HKY + G for the COI mtDNA dataset.
Phylogenetic trees were obtained using Bayesian Inference (BI) and Maximum Likelihood (ML). BI was performed using MrBayes 3.2 (Ronquist et al. Reference Ronquist, Teslenko, der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012) on the online platform CIPRES. The Markov chain Monte Carlo (MCMC) was run with 106 generations saving one tree every 100 generations, with a burn-in set to the first 25% of the trees. Only nodes with posterior probabilities (pp) greater than 95% were considered well supported. The ML analyses were run in RAxML (Guindon and Gascuel Reference Guindon and Gascuel2003) at the online platform CIPRES with 1000 bootstrap replicates. Only nodes with bootstrap values greater than 70% were considered well supported. The BI and ML trees were visualized in FigTree v. 1.3.1 software (Rambaut Reference Rambaut2009) and edited in CorelDraw X6.
Pairwise genetic distances among sequences were calculated using the Kimura-2-parameter (K2P) model and a bootstrap procedure with 1,000 replicates in the software MEGA7 version 7.0 (Kimura Reference Kimura1980; Kumar et al. Reference Kumar, Stecher and Tamura2016).
Results
Morphological description
Allocreadiidae Looss, 1902
Creptotrema Travassos, Artigas & Pereira, Reference Travassos, Artigas and Pereira 1928
Creptotrema cruste n. sp. Alcantara, Ebert, Ferreira-Silva, Pérez-Ponce de León & Silva (Figures 1–3)
Figure 1. Creptotrema cruste n. sp. (Holotype) from the anuran-host Crossodactylus schmidti Gallardo, Reference Gallardo1961. A. Total view. B. Ventral view, with detail of a discrete single ventrolateral muscular lobe on either side of the oral sucker, with distribution of papillae over the oral sucker. C. Detail of the cirrus-sac in ventral view. Scale bar: 500 μm (A), 100 μm (B), and 200 μm (C).
Figure 2. Anterior region of Creptotrema cruste n. sp., highlighting the bivalve shell-shaped musculature associated with the ventral sucker opening. A) General view. B) Detail of the musculature of the ventral sucker opening. VS, ventral sucker; CS, cirrus sac; MVSO, musculature associated with the ventral sucker opening; OS, oral sucker; P, pharynx; VF, vitelline follicles. Scale bar: 100 μm (A) and 50 μm (B).
Figure 3. Detail of the Mehlis’ gland region of Creptotrema cruste n. sp. CS, cirrus sac; LVD, left vitelline duct; MG, Mehlis’ gland; O, ovary; RVD, right vitelline duct; SR, seminal receptacle; VC, vitelline cells. Note the nucleus and the cluster of shell protein globules; VF, vitelline follicles. Scale bar: 50 μm.
(Based on seven adult specimens). Body elongated, 2,240 (1,950–2,860) long, 559 (436–688) wide. Oral sucker subterminal, subspherical, 209 (204–241) long, 214 (182–215) wide, with a single muscular lobe on either side of oral sucker (‘auricles’), stretching from ventral side to lateral area. Oral sucker with 4 inner and 6 outer papillae. Two other papillae anteriorly to auricles. Six pairs of papilla on anterior border, close to oral sucker. Inconspicouous pre-pharynx. Pharynx muscular, subspherical, 89 (89–118) long, 99 (99–128) wide. Oesophagus, 88 (88–162) long. Intestinal caeca extending to posterior body end. Ventral sucker pre-equatorial, 358 (355–406) long, 348 (275–394) wide, narrow and elongated opening, with two bivalve shell-like muscular structures. Genital pore close to caecal bifurcation, anterior to ventral sucker. Ratio oral sucker length to ventral sucker length 1:0.6 (0.5–0.7); ratio oral sucker width to ventral sucker width 1:0.6 (0.5–0.7). Testes two, rounded, in tandem, anterior testis 231 (151–291) long, 204 (172–248) wide; posterior testis 222 (210–276) long, 211 (185–301) wide, juxtaposed next to each other, intercecal. Cirrus-sac well developed, 768 (577–768) long, 59 (55–69) wide, sinuous, passing posterior to ventral sucker, reaching ovary region posteriorly, enclosing seminal vesicle, and unarmed and eversible cirrus. Prostate glands about 25% of cirrus sac length. Pars prostatica not observed. Ovary posterior to ventral sucker, pretesticular, slightly sinistral, obliquely oval to irregular in shape, with entire margin, 180 (143–234) long, 213 (142–213) wide. Vitelline follicles marginal, large, extra- and intra-cecal, not overlapping gonads, extending from pharynx level to posterior end of body, completely separated into two lateral fields but confluent in post-testicular region. Mehlis’ gland close to ovary. Laurer’s canal not observed. Uterus pretesticular, intra-cecal. Eggs operculate, (53–63) long, (35–40) wide. Excretory pore terminal; excretory vesicle I-shaped, reaching anterior testis.
Taxonomic summary
Type host: Crossodactylus schmidti Gallardo, Reference Gallardo1961.
Type locality: Parque Nacional do Iguaçu, municipality of Céu Azul, Paraná state, Brazil (25°9’4.036"S, 53°50’28.777"W).
Other localities: Parque Estadual Rio Guarani, municipality of Três Barras do Paraná, Paraná state (25°26’42.871"S, 53°9’37.879"W) and the municipality of São Miguel do Oeste, Santa Catarina state, Brazil (26°45’36.10"S, 53°31’30.47"W).
Site of infection: small intestine.
Infection level: Nine specimens were found in the small intestines of seven host specimens.
Etymology: The specific epithet is derived from the Latin crusta (= shell) and refers to the presence of a remarkable bivalve shell-shaped musculature at the opening of the ventral sucker.
Remarks
Creptotrema cruste n. sp. is morphologically distinguished from its congeners mainly by the presence of a bivalve shell-shaped musculature at the opening of the ventral sucker. This structure was never reported before in Creptotrema spp., making it a specific differential character.
Morphologically, Creptotrema cruste n. sp. differs further from C. creptotrema, C. foliaceum, C. guacurarii, C. lamothei, C. lynchi, C. macrorchis, C. megacetabulare, C. pati, C. platense, C. schubarti, C. stenopteri, and C. totonacapanense (Razo-Mendivil, Mendoza-Garfias, Pérez-Ponce de León & Rubio-Godoy, Reference Razo-Mendivil, Mendoza-Garfias, Pérez-Ponce de León and Rubio-Godoy2014a) by having two lateral vitelline fields that extend along the body and are confluent at the posterior end, filling the entire posterior end, while these species have two lateral vitelline fields that extend to the posterior end of the body but are not confluent. Creptotrema cruste n. sp. also differs from C. diagonale, C. ocloye n. comb., C. sucumbiosa, and C. tica (Hernández Mena, Pinacho-Pinacho, García-Varela, Mendoza-Garfias & Pérez-Ponce de León, Reference Hernández-Mena, Pinacho-Pinacho, García-Varela, Mendoza-Garfias and Pérez-Ponce de León2019) by having testes in tandem, while these species have oblique testes. The new species differs from C. lobatum (Hernández-Mena, Lynggaard, Mendoza-Garfias & Pérez-Ponce de León, Reference Hernández-Mena, Lynggaard, Mendoza-Garfias and Pérez-Ponce de León2016) by presenting testes with a smooth surface, while C. lobatum possesses lobated testes. Creptotrema cruste n. sp. differs from C. astyanace (Scholz, Aguirre-Macedo & Choudhury, Reference Scholz, Aguirre-Macedo and Choudhury2004), C. conconae, C. foliaceum, and C. paraense by presenting a ventral sucker much larger than the oral sucker, while in C. astyanace and C. conconae, the oral and ventral suckers are of similar sizes, and in C. foliaceum and C. paraense, the oral sucker is much larger than the ventral sucker. Furthermore, the new species exhibits morphometric differences with respect to those observed in other Creptotrema species (Table 1).
Table 1. Summary of morphometrics for the most useful characteristics distinguishing species of Creptotrema. The morphological measurements were reported in micrometers. Measurements are sourced from original descriptions plus selected redescriptions
Interestingly, a similar bivalve shell-shaped musculature at the opening of the ventral sucker was reported for Australotrema brisbanense Khalil, Reference Khalil1981, a trematode parasite of the intestine of the pinkete mullet, Trachystoma petardi (Castlenau, 1875) from Brisbane River, Australia (Khalil Reference Khalil1981). In the description, the author pointed out that the ventral sucker of their specimens presented a ‘transverse opening guarded by a strong muscular sphincter’. Other than the presence of this structure, Creptotrema cruste n. sp. and A. brisbanense are very different, as they belong to Allocreadiidae and Cladorchiidae Fischoeder, 1901, respectively.
Phylogenetic analyses
Two partial sequences of the 28S rDNA gene (GenBank accession numbers OR557501 and OR557502) and one partial sequence of the COI mtDNA gene (GenBank accession number OR552537) of Creptotrema cruste n. sp. were obtained. The two newly generated 28S rDNA sequences of Creptotrema cruste n. sp. were each 1,254 and 1,253 bp in length and, after trimming the ends to the shortest sequence, the final alignment was 1,037 bp long. The ML and BI analyses of the partial 28S rDNA alignment produced phylograms with consistent topologies, most nodes were highly supported (Figure 4). Both analyses recovered Allocreadiidae as a monophyletic group. The newly generated sequences of Creptotrema cruste n. sp. grouped together with all the other sequences of Creptotrema in a monophyletic clade (except for Creptotrema funduli Mueller, Reference Mueller1934) with Wallinia spp. recovered as its sister group. For the partial 28S rDNA, the interspecific genetic divergences found among the sequences of Creptotrema cruste n. sp and Creptotrema spp. varied from 2.0% (C. astyanace) to 4.2% (C. guacurarii).
Figure 4. Bayesian inference phylogram based on 28S rDNA sequences of Allocreadiidae, showing the phylogenetic position of the new species, Creptotrema cruste n. sp. from the Schmidt’s Spinythumb frog Crossodactylus schmidti Gallardo, Reference Gallardo1961. Branch length scale bar indicates the number of substitutions per site. The new sequences are highlighted in bold.
The newly generated COI mtDNA sequence of Creptotrema cruste n. sp. was 446 bp in length, and the final alignment was 351 bp long after trimmed to the shortest sequence. The ML and BI analyses of the partial COI mtDNA alignment recovered identical phylograms with most clades well supported (Figure 5). Both analyses also recovered the sequences of Allocreadiidae as a monophyletic group (i.e., Creptotrema spp., Margotrema spp., Wallinia chavarriae Choudhury, Daverdin & Brooks, Reference Choudhury, Daverdin and Brooks2002, and Allocreadium lobatum Wallin, Reference Wallin1909). The newly generated sequences of Creptotrema cruste n. sp. grouped together with all the other sequences of Creptotrema for which sequences are available in a well-supported monophyletic clade, with Wallinia spp. also recovered as its sister group. For the COI mtDNA gene, the interspecific genetic divergences among the new species and Creptotrema spp. were very high and varied from 15.1% (C. megacetabulare) to 16.8% (C. conconae). Furthermore, in both phylograms, C. ocloye n. comb. was recovered as a member of Creptotrema with high nodal support values.
Figure 5. Bayesian inference phylogram based on COI mtDNA sequences of Allocreadiidae, showing the phylogenetic position of the new species, Creptotrema cruste n. sp. from the Schmidt’s Spinythumb frog Crossodactylus schmidti Gallardo, Reference Gallardo1961. Branch length scale bar indicates the number of substitutions per site. The new sequence is highlighted in bold.
Discussion
The integrative taxonomic approach employed in this study provided robust validation for the recognition of a new species of Creptotrema parasitizing the near-threatened anuran C. schmidti in Brazil. Creptotrema cruste n. sp. is the 17th species known from South America, the second species of the genus found parasitizing an anuran, and the fourth species of allocreadiid parasitizing amphibians. Creptotrema lynchi was first described from the toad Rhinella marina in Colombia (Brooks Reference Brooks1976). After that, some reports on C. lynchi parasitizing fishes were published (Kohn et al. Reference Kohn, Fernandes, Macedo and Abramson1985; Lunaschi and Sutton Reference Lunaschi and Sutton1995; Curran et al. Reference Curran2008). However, Franceschini et al. (Reference Franceschini, Aguiar, Zago, Yamada, Ebert and Silva2021) demonstrated that the individuals identified as C. lynchi in fishes from South America were, in fact, juvenile forms of C. creptotrema, and after studying voucher specimens, these authors proposed the synonymy of these records infecting fishes with C. creptotrema. Despite that, C. lynchi remained as the only valid species of the genus Creptotrema reported from an amphibian in South America. Still, the absence of subsequent reports of C. lynchi from anurans led Curran (Reference Curran2008) to suggest that the toad R. marina may have been an accidental host for the species. Therefore, our results not only formally describe a new Creptotrema species but extend the knowledge on host species for the genus and confirm anurans and fishes as hosts of Creptotrema spp. We also provide the first molecular data of a Creptotrema species parasitizing an anuran host, since C. lynchi still lacks molecular information. As most of the definitive hosts of Creptotrema spp. are preferentially freshwater fishes, it would be necessary to generate new molecular data on C. lynchi to compare with Creptotrema cruste n. sp. and test hypotheses of host specificity of these two species.
Additionally, Freitas (Reference Freitas1960) erected a new genus, Maicuru Freitas, 1900, to accommodate Maicuru solitarium Freitas, Reference Freitas1960, an allocreadiid parasite of Rhinella granulosa (Spix, 1824) in Brazil. However, Caira and Bogea (Reference Caira, Bogea, Jones, Bray and Gibson2005) considered that, although the species present muscular lobes associated with the lateral margins of the oral sucker, the only species in the genus lacks an oesophagus and has bilobed instead of smooth testes, concluding that this taxon should be considered as incertae sedis. However, Curran et al. (Reference Curran, Tkach and Overstreet2011) examined photomicrographs of the holotype of M. solitarium deposited at the Helminthological Collection of the Oswaldo Cruz Institute, Rio de Janeiro, Brazil (CHIOC No. 26646) and concluded that the holotype exhibits a suite of features that strongly confirm this digenean as a member of the family Allocreadiidae. Despite that, molecular studies of M. solitarium are necessary to prove its identity as an allocreadiid.
Furthermore, another allocreadiid trematode, Caudouterina rhyacotritoni Martin, Reference Martin1966, was described as a parasite of the Olympic salamander (Rhyacotriton olympicus [Gaige, 1917]) in western Oregon, USA (Martin Reference Martin1966). The species has not been reported ever since. While describing a new genus and species of trematode, Parabrachycoelium longicaecum Pérez-Ponce de León, Mendoza-Garfias, Razo-Mendivil, and Parra-Olea, Reference Pérez-Ponce de León, Mendoza-Garfias, Razo-Mendivil and Parra-Olea2011, parasitizing a salamander from cloud forests of Veracruz, Mexico, the authors observed a remarkable resemblance with C. rhyacotritoni with the exception of the presence of a spined tegument in P. longicaecum (Pérez-Ponce de León et al. Reference Pérez-Ponce de León, Mendoza-Garfias, Razo-Mendivil and Parra-Olea2011). The species was assigned to the family Brachycoeliidae Looss, 1899. After examining the type-material, the authors confirmed that C. rhyacotritoni does not possess spines in the tegument. It is also necessary to obtain DNA sequences of specimens from the type locality to test the validity of the species parasitizing the Olympic salamander.
Our phylogenetic results are in agreement with previous topologies, which resolved the genus Creptotrema as monophyletic (Francheschini et al. 2021), with the new species placed as the early divergent species of all other Creptotrema spp. Even though C. funduli, a Nearctic species, was described as belonging to Creptotrema, molecular evidence unequivocally has shown that the species does not belong in the new concept of the genus (Francheschini et al. 2021), in the same way Creptotrema agonostomi Salgado-Maldonado, Cabañas-Carranza & Caspeta-Mandujano, Reference Salgado-Maldonado, Cabañas-Carranza and Caspeta-Mandujano1998 from the Mountain Mullet in Mexico was shown not to belong in Creptotrema (Pérez-Ponce de León et al. Reference Pérez-Ponce de León, Sereno-Uribe, García-Varela, Mendoza-Garfias, Hernández-Mena, Pinacho-Pinacho and Choudhury2020). Actually, these authors suggested that a new genus was necessary to accommodate the species occurring in the Nearctic, while Franceschini et al. (Reference Franceschini, Aguiar, Zago, Yamada, Ebert and Silva2021) proposed the species as species inquirenda.
The lowest 28S rDNA interspecific genetic divergence found between Creptotrema cruste n. sp. and its congeners was 2.0% (C. astyanace). Franceschini et al. (Reference Franceschini, Aguiar, Zago, Yamada, Ebert and Silva2021) found lower 28S rDNA interspecific genetic divergence between sequences of C. schubarti and C. guacurarii (0.4%), although the divergence value was accepted by those authors as evidence for differentiating between these species, along with meticulous morphological examination. Although it is known that the 28S rRNA gene is a conserved gene and is not an ideal molecular marker for the delimitation of species, we consider our values of interspecific divergence as strong evidence to support the erection of Creptotrema cruste n. sp. The 28S rRNA gene remains the basis of the species-based molecular classification scheme of trematodes thus far (Pérez-Ponce de León et al. Reference Pérez-Ponce de León and DI2019). This output is also concordant with the phylogeny based on the COI mtDNA, in which the lowest interspecific genetic divergences found between Creptotrema cruste n. sp. and its congeners was 15.1% (C. megacetabulare). Franceschini et al. (Reference Franceschini, Aguiar, Zago, Yamada, Ebert and Silva2021) found COI mtDNA genetic divergences between Creptotrema spp. varying from 6.6 to 16.4%, which is also in agreement with our results. Previous studies have suggested that trematodes show maximum intraspecific divergence up to 2% for mtDNA to be considered a single species (Vilas et al. Reference Vilas, Criscione and Blouin2005). This is considerably below the observed values for the Creptotrema species analyzed here. Therefore, the COI mtDNA interspecific values yielded in this study strongly validate Creptotrema cruste n. sp. as a new species.
Recently, a revised diagnosis of Creptotrema was proposed by Franceschini et al. (Reference Franceschini, Aguiar, Zago, Yamada, Ebert and Silva2021). By using molecular evidence, those authors identified the genus Auriculostoma as a synonym of Creptotrema, reallocating all described species of the former Auriculostoma as members of Creptotrema. Therefore, considering the new diagnosis of Creptotrema proposed by Franceschini et al. (Reference Franceschini, Aguiar, Zago, Yamada, Ebert and Silva2021), A. ocloya (Liquin et al. Reference Liquin, Gilardoni, Cremonte, Saravia, Cristóbal and Davies2022), which was described after the new revision of the genus, should now be considered as Creptotrema ocloye n. comb.
The current knowledge of parasites associated with amphibians is still limited, and further parasitological surveys are needed to increase our understanding of the helminth diversity and their relationships with this host group. Reducing taxonomic gaps is a challenge that implies the parasitological exploration of more host species and the use of standard methods for comparative morphological and molecular analyses. Our results contribute substantially to the progress of the global parasite taxonomy, improve the understanding of the phylogenetic relationships of allocreadiid trematodes, and expand the knowledge on the interaction network of parasite species with amphibian hosts in South America.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X24000099.
Acknowledgements
The following authors would like to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for providing research fellowships: EPA (141322/2018-7), CFS (#150125/2023-2) and thank Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP (#FC3-0198-00006.01.00/22) for research funding, DHM (316264/2021-0), LRF (150041/2017-9), and RJS (311635-2021-0). MBE thanks the UNESP Pro-Rectory of Research (PROPG/PROPe - 04/2022) and São Paulo Research Foundation (FAPESP - 2021/12779-9), RJS and GPPL were also supported by CAPES/PRINT (#88887.839573/2023-00 and #88887.839159/2023-00, respectively).
Competing interest
The authors declare no competing interests with anyone.
Ethical standard
All applicable institutional, national, and international guidelines for the ethical handling of animals and collection of zoological material were followed (SISBio #61940), including recommendations from the Ethics Committee for Animal Experimentation (CEUA-UNESP #1061). According to Brazilian laws, species registration for scientific research purposes was carried out at SisGen (#A5DB3BE).
