Assessing the diversity of freshwater fish trematodes from Laguna Escondida, Los Tuxtlas tropical rainforest, Mexico, using morphology and 28S rDNA sequences as barcodes

Abstract

Despite a great effort made for almost 90 years, the diversity of freshwater fish trematodes in Mexico is still far from being fully known. The addition of molecular data to the description of trematode diversity in the last two decades added the potential to establish more robust species limits and a more accurate biodiversity estimation, but also led in some instances to the recognition of cryptic species complexes. Here, we used sequences of the large subunit of the nuclear ribosomal gene (28S rRNA) as barcodes, and morphological data, to assess the diversity of freshwater fish trematodes from a lake within a tropical rainforest. Eighty freshwater fish specimens of eight species were studied, and 120 trematode specimens were collected. Morphologically, specimens were allocated into nine genera; molecular phylogenetic analyses along with sequence divergence data provided evidence for recognising 11 trematode taxa, six adults and five metacercariae; six of them were identified to species level. Geographical distribution and host association patterns are briefly discussed for each trematode taxa.

Introduction

Mexico is considered a megadiverse country due to its position between the Nearctic and Neotropical biogeographical regions, resulting in a great variety of environments (Morrone et al. 1999). Among vertebrates, fish are the most species rich group with 2,763 species representing near 10% of the total number of known species in the world (Espinosa-Pérez 2014). Freshwater fish are highly diverse, with about 500 species (Lyons et al. 2020), with a high percentage of them as endemic components (Miller et al. 2005). Their helminth fauna has been intensively studied (Pérez-Ponce de León and Choudhury 2005; Scholz and Choudhury 2014). A recent study on the fish–helminth interactions of continental waters of Mexico reported 483 helminth taxa parasitising 371 fish species in 1,070 localities throughout Mexico (García-Prieto et al. 2022). Other studies have shown that some river basins of southeastern Mexico possess higher species richness and endemism levels of freshwater fish helminths (Pérez-Ponce de León and Choudhury 2005; Aguilar-Aguilar et al. 2008; Pérez-Ponce de León et al. 2011). Still, most records lack genetic information associated to the species of helminth infecting freshwater fishes in Mexico.

Los Tuxtlas tropical rain forest is located in southeastern Mexico, in the coastal plain of the Gulf of Mexico (Von Thaden et al. 2020). Laguna Escondida (LE) is a relatively small freshwater reservoir within Los Tuxtlas region; thirteen freshwater fish species have been reported in the locality from the orders Atheriniformes: Atherinella alvarezi (Regan); Cichliformes: Rocio octofasciata (Regan), Vieja fenestrata (Günther); Characiformes: Astyanax aeneus (Günther), and Astyanax finitimus Bocourt; Cyprinodontiformes: Belonesox belizanus Kner, Gambusia sexradiata Hubbs, Heterandria bimaculata (Heckel), Poecilia mexicana Steindachner, and Xiphophorus helleri (Heckel); Mugiliformes: Dajaus monticola (Bancroft); Siluriformes: Rhamdia guatemalensis (Gunther), and Cathorops aguadulce (Meek) (Espinosa-Pérez 2017). Irrespective of the large fish diversity, information about their parasite fauna is scarce (see Salgado-Maldonado et al. 2005), and no assessment of the genetic diversity has been made. The trematode genetic diversity of LE has been only assessed through molecular studies of cercariae released from mollusks looking for the link between these larval forms and metacercariae with adults for elucidating parasite life cycles (see Velázquez-Urrieta and Pérez-Ponce de León 2020, 2021).

Considering that fish act either as intermediate or definitive hosts of many trematode species, the main objective of this study is to uncover the diversity of trematodes in freshwater fish from LE using morphology and 28S rDNA sequences to assess the identity, genetic diversity, and phylogenetic position of adults and metacercariae.

Materials and methods

Specimens collection and morphological analyses

Specimens of freshwater fish were collected in Laguna Escondida (18°38’09”N, 95°07’28”W) in January 2019 and 2020. Fish were captured using cast nets, kept alive in containers with water from the collecting site, and transported to the laboratory. Fish euthanisation was carried out in strict accordance with the American Veterinary Medical Association Guidelines for the Euthanasia of Animals: 2020 edition (https//www.avma.org/sites/default/files/2020-02/Guidelines-on-Euthanasia-2020.pdf) and immediately examined for ecto- and endoparasites. Internal organs were separated in Petri dishes with 0.65% saline and analysed under the stereomicroscope. For morphological analysis, specimens were fixed in 4% hot formalin and preserved in ethanol (70%). Some individuals were stained with Gomori’s trichrome, dehydrated through ethanol series, cleared in methyl salicylate, and mounted in Canada balsam. Voucher specimens were deposited in the Colección Nacional de Helmintos, Instituto de Biología, UNAM (CNHE). For molecular analyses, worms were preserved in 100% ethanol.

Molecular analyses

For molecular analyses, total DNA was extracted from each individual worm using DNAzol, following the protocol provided by the company (Molecular Research Canter, USA) (Chomczynski et al. 1997). Amplification and sequencing of the 28S rRNA gene was carried out using the primes: 28SL 5′ -AAC AGT GCG TGA AAC CGC CTC-3′ (Palumbi 1996) and LO 5′ -GCT ATC CTG AGR GAA ACT TCG-3′ (Tkach et al. 2000). Thermal cycling conditions for amplification reactions were 94°C for 1 min, followed by 35 cycles at 92°C for 45 s, 50°C for 40 min, 72°C for 1 min, and a final extension at 72°C for 10 min. Sequencing reactions were accomplished using an ABI 3730xl Genetic Analyzer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at the Laboratorio Nacional de Biodiversidad, Instituto de Biología. Universidad Nacional Autónoma de México (LANABIO).

Sequences were edited and assembled using the program Geneious 5.1.7 (Biomatters Ltd. Auckland, New Zealand). All alignments were trimmed to the shortest sequence. The length of the alignment was variable among the analysed taxa. The sequences were first screened thorough the BLAST search (GenBank) for assigning them into the lowest taxonomic level through sequence identity values. Once allocated into family, newly generated sequences were aligned separately with some representative sequences downloaded from GenBank for species allocated in the families Allocreadiidae, Apocreadiidae, Clinostomidae, Cryptogonimidae, Derogenidae, Diplostomidae, Gorgoderidae, Haploporidae, and Heterophyidae; then, phylogenetic analyses were conducted separately. Alignments were built, and the number of base pairs is variable since each alignment was trimmed to the shortest sequence. DNA sequences were aligned using MAFFT (Katoh and Standley 2013), with the default parameters. Uncorrected p distances were obtained in MEGA-X (Kumar et al. 2018). Phylogenetic analyses were performed through Bayesian inference (BI), using Markov Chain Monte Carlo (MCMC) in Mr. Bayes V 3.1.2 (Ronquist et al. 2012), with appropriate model of evolution GTR+1+Γ determined with jModeltest 0.1.1 (Posada 2008). The chains were run for 1,500,000 generations, sampling trees every 1,000 generations; the first 25% of the sampled trees were discarded according to Tracer V 1.5 (htt://beast.bio.ed.ac.uk/ tracer); consensus topology and posterior probability values were calculated from the remaining 75% of the trees.

Results and discussion

Eighty individuals of freshwater fish from eight species were examined for trematodes (Table 1); seventy-three of the 80 analysed hosts were infected with at least one species of trematode. In total, 120 trematode specimens were collected. Morphologically, specimens were allocated into nine genera – namely, Ascocotyle Looss, 1899; Clinostomum Leidy, 1856; Crassicutis Manter, 1936; Creptotrematina Yamaguti, 1954; Genarchella Travassos, Artigas & Pereira, 1928; Oligogonotylus Watson, 1976; Saccocoelioides Szidat, 1954; Posthodiplostomum Dubois, 1936; and Phyllodistomum Braun, 1899. The preliminary nBLAST search of 28S rDNA corroborated the identification to genus level. In most instances, nBLAST search also confirmed conspecificity through a sequence identity higher than 99%, and individuals were identified up to species level. Morphology and 28S rDNA sequence data recognised 11 trematode taxa infecting fish from Laguna Escondida; six of them were identified to species level (Table 1, Figure 1). Of the 11 trematode taxa, seven were adults, and four metacercariae (Table 1). The fish species with the largest trematode diversity was the poecilid Ga. sexradiata, with six taxa (Table 1). Along with the nBLAST search, phylogenetic analyses were run through Bayesian inference to test the position of the sequenced individuals in the trematode phylogenetic tree, considering the families where each species is allocated (Figures 2–4).

Table 1. Species of trematodes found in freshwater fishes of Laguna Escondida, Los Tuxtlas tropical rainforest, ordered alphabetically by family

Figure 1. Line drawings of the trematodes of freshwater fishes from Laguna Escondida, Los Tuxtlas, Veracruz. Clinostomum sp. (a); Posthodiplostomum sp. 1 (b); C. cichlasomae (c); C. aguirrepequenoi (d); G. astyanactis (e); O. manteri (f); P. inecoli (g); S. orosiensis (h).

Figure 2. Bayesian phylogenetic trees of the 28S rDNA showing the phylogenetic positions of trematodes sequences from Laguna Escondida, Los Tuxtlas. Ascocotyle spp. (a); Clinostomum spp. (b); and Posthodiplostomum spp. (c).

The following species/taxa were identified (presented by developmental stage and ordered alphabetically):

Metacercariae

Ascocotyle sp.

Metacercariae from the mesenteries of V. fenestrata and G. sexradiata were allocated to the genus Ascocotyle. The newly sequenced isolates were aligned with 28 sequences of Ascocotyle spp. Alignment was 967 base pair long and recovered the sequences from Laguna Escondida within a clade containing isolates of Ascocotyle from the same locality in Veracruz (sequenced from cercarial stages released from snails; Velázquez-Urrieta and Pérez-Ponce de León 2021), and from cichlids of Lake Nicaragua (Santacruz et al. 2022 erroneously identified it as A. pindoramensis (Travassos, 1928)), and varied from these sequences only between 0 and 0.55%, indicating they all represent the same species (Figure 2a). At least 14 species of Ascocotyle have been reported in Mexico, most of them as larval stages in freshwater and estuarine habitats (Pérez-Ponce de León et al. 2007; Espínola-Novelo et al. 2023), although no sequence data has been generated for adult forms yet to match with the larval forms from snails and fish. In Veracruz, where the tropical rainforest of Los Tuxtlas lies, six species of Ascocotyle have been reported (see Velázquez-Urrieta and Pérez-Ponce de León 2021). The newly sequenced individuals match one of the sequences obtained from snails in Laguna Escondida, although no adults have been found in birds to confirm species identity. Two species of Ascocotyle have been reported previously in Laguna Escondida: A. tenuicollis Price, 1935 (= A. felippei Travassos, 1928) from A. aeneus and from X. helleri, and A. nana Ransom, 1920 from X. helleri (Salgado-Maldonado et al. 2005). Both species are widely distributed in Mexico; metacercariae of A. tenuicollis have been reported from 24 freshwater fish species, and adults from four species of fish-eating birds, whereas A. nana has been found in 23 freshwater fish species and adults in three species (see Scholz et al. 2001 and references therein). The lack of sequence data for adults of Ascocotyle prevents establishing a link between larval forms and adults to identify the species; unfortunately, very few specimens were recovered from their hosts, and the quality of the material is very poor to achieve the identification following Scholz et al. (2001) identification key for larval stages since the number of spines was not established.

Clinostomum sp.

Metacercariae encysted in the mesenteries, gill arches, and fins of D. monticola, A. aeneus, H. bimaculata, B. belizanus, and P. mexicana were morphologically identified as belonging to Clinostomum. These metacercariae are characterised by having a yellow colour, an elongate body, a well-developed and characteristic oral collar, small pharynx, and the genital organs scarcely developed (Figure 1a). The phylogenetic analyses included 11 newly generated sequences, and 10 additional sequences for Clinostomum spp. available in GenBan; the alignment was 980 bp long. The phylogenetic tree showed that the new sequences from Los Tuxtlas nested with Clinostomum sp. from an ardeid from Catemaco Lake, Veracruz (MH159733) (Figure 2b), a locality close to Laguna Escondida, although with a genetic divergence varying between 1.0% and 1.8%. In the case of Clinostomum, molecular markers other than 28S rDNA such as cytochrome c oxidase subunit 1 (cox1) and the internal transcribed spacers are better loci for species identification since the genetic library is more complete (see Locke et al. 2015a; Pérez-Ponce de León et al. 2016). Sereno-Uribe et al. (2022) showed that cox1 is the most appropriate marker for species delimitation for species in the genus. Still, novel 28S sequences of Clinostomum obtained in our study from five species of freshwater fishes show they are almost identical. Pérez-Ponce de León et al. (2016) identified at least three putative species of Clinostomum from samples obtained from Catemaco Lake. Two of them were described as Clinostomum caffarae Sereno-Uribe, García-Varela, Pinacho-Pinacho & Pérez-Ponce de León, 2018 and Clinostomum arquus Sereno-Uribe, García-Varela, Pinacho-Pinacho & Pérez-Ponce de León, 2018 based on adults sampled from the snowy egret, Egretta thula Molina. However, no 28S rDNA sequences were generated for these species, and we cannot compare them at this time. Clinostomum complanatum (Rudolphi, 1814) was previously reported by Salgado-Maldonado et al. (2005) as a parasite of P. mexicana; however, this represents a misidentification by the authors since it has been proven that C. complanatum is not distributed in the Americas (Pérez-Ponce de León et al. 2016). The identification of this larval form at species level is pending until DNA sequences are obtained for other molecular markers.

Posthodiplostomum spp.

Encysted metacercariae from the mesenteries of H. bimaculata, A. aeneus, G. sexradiata, and V. fenestratum were identified as belonging to the genus Posthodiplostomum; they are mainly characterised by having a body divided in two segments, a well-developed tribocytic organ in the anterior segment, and gonads poorly developed in the posterior segment (Figure 1b, Posthodiplostomum sp.1). Six isolates of the newly sequenced specimens were aligned with 25 sequences of Posthodiplostomum spp. available in GenBank for the 28S rRNA gene. Alignment was 1,015 bp. The phylogenetic tree yielded sequences from Laguna Escondida in two reciprocally monophyletic clades, showing they represent two separate species (Figure 2c). At this time, 28S rDNA sequences are only useful to recognise two separate lineages of Posthodiplostomum in Laguna Escondida, although none of them correspond to a previously described species. The nBLAST search did not yield a match with a previously described species. As in the case of Clinostomum, the genetic library for species of Posthodiplostomum is increasing steadily, and other molecular markers as cox1 seem to be more accurate for species delimitation (see Locke et al. 2015b; Pérez-Ponce de León et al. 2022). Morphologically, metacercarial stages cannot be separated, and this represents a practical problem that can only be overcome by using DNA sequences. In a previous study, Salgado-Malonado et al. (2005) reported P. minimum (McCallum, 1921) parasitising V. fenestrata in Laguna Escondida; however, molecular analyses have shown that the genus Posthodiplostomum is constituted by at least six genetic lineages infecting freshwater fishes of Mexico, and none of them represents the species P. minimum. Our study revealed that V. fenestrata is, in fact, infected with two genetic lineages of Posthodiplostomum in Laguna Escondida, and neither of them correspond to P. minimum.

Adults

Crassicutis cichlasomae Manter, 1936

Trematodes from the intestines of D. monticola and P. mexicana were morphologically identified as C. cichlasomae because they have an oval body, pretesticular uterus, and a body surface without spines (Figure 1c); they were, however, juveniles. Due to the unusual host association (the species is a cichlid specialist, see Choudhury et al. 2017), identification was corroborated through 28S rDNA sequences. Four newly sequenced isolates were aligned with 13 sequences of four species of Crassicutis available in GenBank. Alignment was 999 bp long. The phylogenetic tree showed that the new sequences nested in a moderately supported clade with C. cichlasomae from cichlid fish of Mexico; sequence divergence was null, showing conspecificity, and appeared as the sister taxa of C. choudhuryi Perez-Ponce de León, Razo-Mendivil, Rosas, Mendoza & Mejia, 2008 from cichlids of Nayarit Mexico, with a divergence value of 1.3% (Figure 3a). Species of Crassicutis are members of the core parasite fauna of cichlids (sensu Pérez-Ponce de León and Choudhury 2005). For instance, Pérez-Ponce de León et al. (2007) reported C. cichlasomae in 21 species of cichlids belonging to eight genera across the country. Since we sampled juvenile individuals from the intestine of a mugilid (D. monticola) and a poecilid (P. mexicana), we consider those as accidental infections. Both fish species are omnivorous and may have fed upon snails. Snails of the genus Pyrgophorus coronatus act as first and second intermediate host of Crassicutis (Scholz et al. 1995); most likely, even though D. monticola and P. mexicana were infected, the parasite did not reach sexual maturity. We did not sample cichlids in our study, but V. fenestrata occurs in the locality (Espinosa-Pérez 2017), and, in fact, C. cichlasomae was previously reported from that cichlid species in Laguna Escondida by Salgado-Maldonado et al. (2005).

Figure 3. Bayesian phylogenetic trees of the 28S rDNA showing the phylogenetic positions of trematodes sequences from Laguna Escondida, Los Tuxtlas. Crassicutis spp. (a); Creptotrematina (b); and Genarchella spp. (c).

Creptotrematina aguirrepequenoi Jiménez Guzmán, 1973

Trematodes from intestines of A. aeneus were morphologically very similar to C. aguirrepequenoi, which are characterised by the presence of an elongated body, ventral sucker larger than the oral sucker, large number of small eggs, the uterus extending to the posterior end of the body, and the oral sucker with a pair of ventrolateral muscular lobes (Figure 1d). The 28S rDNA phylogenetic analysis consisted of 11 sequences of allocreadiids; alignment was 1,044 bp long. The newly generated sequences nested together with those of C. aguirrepequenoi (specimens from Mexico and Costa Rica) with high nodal support; our sequences differed from those in GenBank between 0.1 and 0.2%, indicating conspecificity (Figure 3b). All these sequences nest as the sister taxa of C. batalhensis Dias & Abdhalla, 2020 described from Astyanax spp. in Brazil (Dias et al. 2020). Creptotrematina aguirrepequenoi is apparently widely distributed across Middle America. The species was originally described by Jiménez-Guzman (1973) from Astyanax mexicanus (De Filippi) in northern Mexico, but it has been reported from Astyanax spp. from across Mexico, including Veracruz, but also in Costa Rica (Curran et al. 2011; Razo-Mendivil et al. 2014). A previous record of the species Magnivitellinum simplex Kloss, 1966 as a parasite of A. aeneus in Laguna Escondida was made by Salgado-Maldonado et al (2005). The species we report here is morphologically similar to M. simplex, and we tried to corroborate the identification by studying museum specimens since Salgado-Maldonado et al. (2005) mentioned that specimens were deposited in the Colección Nacional de Helmintos; however, they were actually not deposited, and we could not confirm if that was a misidentification.

Genarchella astyanactis (Watson, 1976)

Specimens from the stomachs of Ga. sexradiata were morphologically identified as G. astyanactis. They are characterised by having an elongated body, curved seminal vesicle and vitellarium formed by two compact masses lying in the posterior end of the body (Figure 1e). The new sequences were aligned with other those of other species of derogenids, hirudinellids and didymozoids; the alignment consisted of 14 sequences and was 1,085 bp long. In the phylogenetic tree the new sequences were nested in a well-supported clade with specimens of G. astyanactis from Yucatán, Mexico and Lake Nicaragua, Nicaragua, and sequences were almost identical, varying 0–0.9%; this clade was recovered as the sister group of G. pichileufuensis Tsuchida, Urabe, Viozzi, Rauque & Flores, 2021 from a siluriform in Argentina, with divergence of 2.6% (Figure 3c) (Tsuchida et al. 2021). Another species of Genarchella was recently described by Moravec & Prouza (2024), G. venezuelensis, as a parasite of a loricariid siluriform in Venezuela, although is clearly differentiated from G. astyanactis by having a distinct median papilla-like lobe the anterior margin of the ventral sucker. The species was originally described from a characid in Lake Nicaragua (Watson, 1976), but has been found in Astyanax spp. in Mexico (Pérez-Ponce de León et al. 2007). Interestingly, the species seems to be specific to characids of the genus Astyanax, but the species was reported here in a poecilid (G. sexradiata). We studied specimens of the characid A. aeneus (Günther) but we were unable to obtain samples of this species. In this case, since adults were sexually mature, we cannot consider it represents an accidental infection.

Oligogonotylus manteri Watson, 1976

Some adults from the intestines of G. sexradiata were identified as belonging to the cryptogonimid genus Oligogonotylus Watson, 1976 by having a longitudinal row of five to nine sucker-like gonotyls, and vitelline follicles extending between the pharynx level and anterior margin of the ovary (Figure 1f). Particularly, they match with the description of O. manteri. The phylogenetic tree included sequences of other cryptogonimids for an alignment of 14 terminals and 1,069 bp (Figure 4a). The newly sequenced individuals nested in a well-supported clade with those of O. manteri from Yucatán, Mexico, and from Lake Nicaragua, with null or very low sequence divergence values (0–0.1%). This species was recovered as the sister taxa of O. mayae Razo-Mendivil, Rosas-Valdez & Pérez-Ponce de León, 2008 from Mexico, with divergence values varying from 1.7% to 1.9%. No sequence data for the 28S rRNA gene were available for comparison for the species O. andinus Vélez-Sampedro, Uruburu & Lenis, 2022 described from poeciliids and cichlids in Colombia (Vélez-Sampedro et al. 2022). Adults of O. manteri have been reported in Mexico from 10 species of cichlids allocated in six genera. The metacercaria has been reported from the eyes, fins, gills, heart, intestine, mesentery, opercula, rectum, scales, and spleen of 10 species of cichlids, but also it has been found in another eight fish species, exhibiting low host specificity at this level (see Pérez-Ponce de León et al. 2007). Still, this represents the first report of adults of the species parasitising the intestine of a poecilid fish. Since all specimens are juvenile, we consider this an accidental infection.

Figure 4. Bayesian phylogenetic trees of the 28S rDNA showing the phylogenetic positions of trematodes sequences from Laguna Escondida, Los Tuxtlas. Oligogonotylus spp. (a); Phyllodistomum spp. (b); Genarchella spp. (c); and Saccocoelioides (d).

Phyllodistomum spp.

The specimens collected from the urinary bladder of the poeciliids X. helleri, B. belizanus, and H. bimaculata were morphologically identified as members of the genus Phyllodistomum; specimens were characterised by having a foliated hindbody, two compact and post-acetabular vitelline masses, and the uterus strongly coiled and extending in the hindbody (Figure 1g). Three individuals were sequenced and aligned with 42 sequences representing 28 species of Phyllodistomum. Alignment was 1,041 bp long; the phylogenetic tree yielded one of the newly generated sequences nested within P. inecoli Razo-Mendivil, Pérez-Ponce de León & Rubio-Godoy, 2013, a parasite of poeciliids from Mexico, with very low genetic divergence (0.1%) indicating conspecificity; two additional sequences were recovered as an independent clade, as the sister group to a clade containing sequences of P. wallacei Pérez-Ponce de León, Martínez-Aquino & Mendoza-Garfias, 2015 and P. spinopapillatum Pérez-Ponce de León, Pinacho-Pinacho, Mendoza-Garfias & García-Varela, 2015, P. simonae Pinacho-Pinacho, Sereno-Uribe, Hernández-Orts, García-Varela & Pérez-Ponce de León, 2021 + P. inecoli Razo-Mendivil, Pérez-Ponce de León & Rubio-Godoy, 2013, albeit with moderate nodal support value (0.89) (Figure 4b). The genetic divergence between the new sequenced individuals and the three species mentioned above varied from 0.6 to 1.5%. In this case, 28S rDNA sequences are not very useful in establishing species limits in a group of species of Phyllodistomum infecting cyprinodontiform fishes (see Pinacho-Pinacho et al. 2021). These authors found a low genetic divergence among the species contained in that clade, varying from 0.1 to 1.4% for the 28S rRNA gene, although estimated divergence of cox1 between the same species varied between 5.1 and 10.7%. Criteria to consider these species as independent units also considered host association and geographical distribution – that is, P. inecoli is a parasite of poeciliids in river basins draining to the Gulf of Mexico coast; P. spinopapillatum is found in profundulids in river basins draining to the Pacific coast, in Oaxaca; P. simonae infect an endemic species of profundulid in a close basin in the highlands of Chiapas; and P. wallacei is found in an endemic goodeid in the Cuzalapa and Ayuqila river basins in Michoacán, which drain to the Pacific coast (Razo-Mendivil et al. 2013; Pérez-Ponce de León et al. 2015a; Pérez-Ponce de León et al. 2015b; Pinacho-Pinacho et al. 2021). The position in the phylogenetic tree and the sequence divergence of the two newly sequenced individuals sampled from X. helleri and B. belizanus from Laguna Escondida suggest they represent an undescribed species. Unfortunately, we sampled very few individuals, preventing the proper description of the new species; furthermore, it would be necessary to obtain cox1 sequences to establish a more robust species delimitation by comparing with sequences of species available in GenBank.

Saccocoelioides orosiensis Curran, Pulis, Andres & Overstreet, 2018

Trematodes from the intestines of D. monticola, A. aeneus, and H. bimaculata were initially allocated in the genus Saccocoelioides by having a small body with the tegument entirely covered by minute spines, dispersed eye-spot remnants in anterior half of the body, and sac-like caeca (Figure 1h); however, identification to species level relied on molecular data since members of the genus are difficult to identify due to their small body size. The 28S rDNA phylogenetic analyses of Saccocoelioides included five newly sequenced isolates and 12 specimens of 11 congeneric species for which 28S rDNA sequences are available; the alignment was 1061 bp long. In the phylogenetic tree, the new sequences nested in a highly supported monophyletic clade with S. orosiensis from poeciliids of Mexico, varying only between 0 and 0.1%, showing conspecificity. The species was yielded nested as the sister species of S. nani Szidat 1954 from Argentina with a divergence of 1.18% (Figure 4c). Saccocoelioides orosiensis was originally described by Curran et al. (2018) as a parasite of the poecilid Poecilia gilli (Kner) in northwestern Costa Rica. Interestingly, Curran et al. (2018) also described a new species of Saccocoelioides from A. aeneus from the same locality in Costa Rica. We sampled specimens from three host species, including the poecilid H. bimaculata, the mugilid D. monticola, and the characid A. aeneus; our sequenced individuals from A. aeneus correspond with S. orosiensis. Also, S. chauhani, which was first described by Lamothe (1974) from Astyanax aeneus from Catemaco Lake (a nearby locality to Laguna Escondida), is also clearly separated in the phylogenetic analysis. Saccocoelioides cf. sogandaresi was reported by Salgado-Maldonado et al. (2005) as a parasite of P. mexicana. Clearly, the authors were not totally convinced about the identification since they recognised that the species was morphologically like S. sogandaresi. In our study, we report S. orosiensis also as a parasite of a poecilid from the same locality based on morphological and molecular data; the record by Salgado-Maldonado et al. (2005) most likely corresponds to the species S. orosiensis.

Final considerations

This study reports 11 taxa of trematodes infecting freshwater fish in a relatively small reservoir (958 by 267 m) enclaved in Los Tuxtlas tropical rainforest, and identifications are validated by using both morphological and molecular data; this reservoir drains to the Río Máquinas, which opens into the Gulf of Mexico. Most of the taxa identified correspond to adult forms (64%); six of the seven adult species have been reported across southeastern Mexico. Some of them, even in other areas of Central America, correspond to a neotropical component which is the result of the distribution of their hosts, primarily cichlids, characids, and poeciliids (Choudhury et al. 2016, 2017). Only four taxa were found as metacercariae; all of them complete their life cycle when fish are consumed by fish-eating birds, primarily herons, and are generalist species in freshwater fishes since they are commonly found in a wide array of fish species (see Scholz et al. 2001; Pérez-Ponce de León et al. 2007).

Our study reinforces the importance of using DNA sequence data to accomplish a more accurate species delimitation and then a better estimation and understanding of trematode diversity. 28S rDNA sequences are useful as barcodes to identify trematodes, although we acknowledge that in some cases, as shown in the genera Clinostomum, Posthodiplostomum, and Phyllodistomum in the present study, it is necessary to consider other molecular markers more variable than 28S rDNA. In the genera mentioned above, the genetic library has been increased in the last years for other markers such as the internal transcribed spacers (ITS1-5.8-ITS2) and the mitochondrial cox1. Still, in our opinion for a general investigation about trematode diversity, sequences of the 28S rRNA gene can be used as an initial step to accomplish the goal. This approach is more important when studies are centered in the first and second intermediate hosts, especially if a molecular link can be established with sequences of adults. The 28S rDNA genetic library of trematodes increases steadily; this is the molecular marker upon which the current classification scheme of trematodes is based on (Olson et al. 2003; Pérez-Ponce de León & Hernández-Mena, 2019). In some cases, a thorough study with other molecular markers, in the context of integrative taxonomy, will be necessary to detect cryptic species complexes through a molecular prospecting approach.