Introduction
The karstic nature of the Yucatán Peninsula creates a unique limnological system, where groundwater represents the only freshwater source in the region (Bauer-Gottwein et al. Reference Bauer-Gottwein, Gondwe, Charvet, Marín, Rebolledo-Vieyra and Merediz-Alonso2011). The Peninsula is characterised by an almost complete lack of inland surface water bodies, with no rivers and only a few small lakes in its southernmost region (Reddell Reference Reddell1977, Schmitter-Soto et al. Reference Schmitter-Soto, Comín, Escobar-Briones, Herrera-Silveira, Alcocer, Suárez-Morales, Elías-Gutiérrez, Díaz-Arce, Marín and Steinich2002); numerous aquatic karstic elements connected to the aquifer, locally known as cenotes (from the Mayan tz’ono’ot), are present (Gaona-Vizcayno et al. Reference Gaona-Vizcayno, Gordillo-de Anda and Villasuso-Pino1980). The geomorphological characteristics of cenotes and their underground connection to the cave systems determine the presence of highly fragile ecosystems which provide habitat for numerous species, some of which are endemic (MacSwiney et al. Reference MacSwiney, Vilchis, Clarke and Racey2007; Moreno-Pérez et al., Reference Moreno-Pérez, Hernández-Téllez, Ramirez-Durán, Gamboa-Angulo and Isaac-Olivé2020). Interestingly, cenotes are aligned and remarkably abundant along a ~200 km semicircle referred to as the ‘Ring of Cenotes’ (RoC), a regional-scale structure in the northwest of the Peninsula overlying the Chicxulub impact crater associated with the Cretaceous-Paleogene (K-Pg) mass extinction (Perry et al. Reference Perry, Marin, McClain and Velazquez1995). It has been estimated that there are 9,000 cenotes in the Yucatán Peninsula (Lara-Lara et al. Reference Lara-Lara, Arreola-Lizárraga, Calderón-Aguilera, Camacho-Ibar, De la Lanza-Espino, Escofet-Giansone, Espejel, Guzmán-Arroyo, Ladah, López-Hernández, Melling-López, Moreno-Casasola, Reyes-Bonilla, Rios-Jara, Zertuche, Soberón, Halfter and Llorente-Bousquets2008). However, the locations of most are imprecise due to difficult access (Moreno-Pérez et al. Reference Moreno-Pérez, Hernández-Téllez, Ramirez-Durán, Gamboa-Angulo and Isaac-Olivé2020). Still, at least 1026 cenotes have been georeferenced, one in Campeche, 42 in Quintana Roo, and 983 in Yucatán. Despite their relevance as the main water source for human settlements, the freshwater fish parasite fauna of only 50 cenotes across the Yucatán Peninsula has been assessed.
The first reports on fish helminth parasite species occurring in cenotes of Yucatán were made by Manter (Reference Manter, Pearse, Creaser and Hall1936), Pearse (Reference Pearse, Pearse, Creaser and Hall1936), Moore (Reference Moore, Pearse, Creaser and Hall1936), and Stunkard (Reference Stunkard1938). Fish parasite surveys were neglected in the area for several decades, until Scholz and co-workers conducted studies on the helminth parasite fauna of cenotes across the Yucatán Peninsula (Mendoza-Franco et al. Reference Mendoza-Franco, Scholz, Vivas-Rodríguez and Vargas-Vázquez1999; Scholz et al. Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Franco1995a, Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Francob, Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Franco1996a; Moravec et al. Reference Moravec, Vivas-Rodriguez, Scholz, Vargas-Vázquez, Mendoza-Franco, Schmitter-Soto and Gonzalez-Solis1995a, Reference Moravec, Vivas-Rodriguez, Scholz, Vargas-Vázquez, Mendoza-Franco, Schmitter-Soto and Gonzalez-Solisb). These authors provided data on the morphology, distribution, host specificity, and life cycle of fish parasites in these environments, and concluded that trematodes, mainly as metacercarial stages, were the dominant group of helminths of cenotes-dwelling fish, with 10 species as adults and 21 species identified as metacercariae (Scholz et al. Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Franco1995a,Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Francob). Subsequent reports on fish parasites from cenotes are scarce and scattered in the literature, although some of these contributions began to generate molecular data (e.g., Pérez-Ponce de León et al. Reference Pérez-Ponce de León, Razo-Mendivil, Rosas-Valdez, Mendoza-Garfias and Mejía-Madrid2008; Razo-Mendivil et al. Reference Razo-Mendivil, Vázquez-Domínguez, Rosas-Valdez, Pérez-Ponce de León and Nadler2010).
The identification of most fish trematode species reported in cenotes was mostly based on morphological characters. Even though very few sequence data have been generated for trematode metacercariae in freshwater fish of cenotes of the Yucatán Peninsula (e.g., Pérez-Ponce de León et al., Reference Pérez-Ponce de León, Razo-Mendivil, Rosas-Valdez, Mendoza-Garfias and Mejía-Madrid2008; Razo-Mendivil et al., Reference Razo-Mendivil, Vázquez-Domínguez, Rosas-Valdez, Pérez-Ponce de León and Nadler2010), we acknowledge that the integration of morphological and molecular data is currently considered essential to determine the identity of trematode species, as recently discussed by Cribb et al. (Reference Cribb, Barton, Blair, Bott, Bray, Corner, Cutmore, De Silva, Duong, Faltýnková, Gonchar, Hechinger, Herrmann, Huston, Johnson, Kremnev, Kuchta, Louvard, Luus-Powell, Martin, Miller, Pérez-Ponce de León, Smit, Tkach, Truter, Waki, Vermaak, Wee, Yong and Achatz2025). This approach allows a more accurate estimation of their diversity and consequently a better understanding of host specificity patterns and life cycles. Recently published studies have addressed this approach for understanding the freshwater fish trematode metacercariae diversity in southern Africa and Europe (Kudlai et al. Reference Kudlai, Binkienė, Rakauskas and Baker2026; Truter et al. Reference Truter, Yong, Smit, Chakona, Luus-Powell and Smit2025). Therefore, the aim of the present study is to describe the diversity of metacercariae parasitising fish of cenotes distributed within the RoC through their morphological and molecular characterisation, to assess the taxonomic identity of these larval forms, and to test their phylogenetic position within the family-level classification scheme of the Digenea.
Materials and methods
Study area and fish sampling
A total of 236 individuals from seven fish species belonging to the families Cichlidae Bonaparte, 1835, Poeciliidae Bonaparte, 1831, or Heptapteridae Gill, 1861 were collected between April 2024 and April 2026 in 15 cenotes within the RoC, in Yucatán State, Mexico (Figure 1, Table 1). Fish were captured using fishing rods, line, and hooks, and foldable fish traps. Caught fish were separated by species in water-filled containers, transported to the laboratory, and kept alive until their examination, within the next 48 h after capture.
Map of the Yucatán Peninsula showing the sampling sites (sinkholes) across the RoC.
Figure 1. Long description
A geographical map with a coordinate grid showing longitude from -90.00 to -88.00 and latitude from 20.00 to 21.00.
* Context: An inset map in the top-left corner shows the location of the Yucatan Peninsula within Mexico.
* Regional Layout: The main map highlights the state of YUCATAN in light pink, bordered by CAMPECHE to the Southwest and QUINTANA ROO to the Southeast. The Gulf of Mexico lies to the North.
* Key Feature: A semi-circular arc, the Ring of Cenotes, is delineated by two parallel dashed gray lines curving from the West coast near Celestun, through the center of the state, and up toward the Northeast coast near Dzilam de Bravo.
* Sampling Sites: 15 black dots representing sinkholes are numbered and positioned within or near this arc.
- Sites 1 and 2 are in the West near Celestun.
- Site 3 is near Kopoma.
- Site 15 is South of Merida.
- Sites 4, 5, 6, 7, 8, 9, and 10 are clustered in the South-Central part of the arc near Tekit.
- Sites 11 and 12 are near Izamal.
- Sites 13 and 14 are in the Northeast near Buctzotz.
* Scale and Orientation: A scale bar in the bottom-right indicates 0, 25, and 50 k m. A North arrow is located at the bottom-right corner.
Sampling sites (cenotes), coordinates and fish collected. Numbers of cenotes correspond to those shown in Fig. 1
Table 1. Long description
The table consists of four columns: Number, Cenote, Coordinates, and Fish species with sample size n.
* Site 1: Sabtún at 20 degrees 51 minutes 0.936 seconds North, 90 degrees 14 minutes 8.124 seconds West. Species: Thorichthys meeki (n = 14).
* Site 2: Noc-Choncunchey at 20 degrees 48 minutes 47.002 seconds North, 90 degrees 11 minutes 48.001 seconds West. Species: Thorichthys meeki (n = 7), Rocio octofasciata (n = 1), Mayaheros urophthalmus (n = 8), and Rhamdia guatemalensis (n = 1).
* Site 3: Chen ha at 20 degrees 41 minutes 22.128 seconds North, 89 degrees 52 minutes 33.203 seconds West. Species: Mayaheros urophthalmus (n = 12), Poecilia velífera (n = 3), and Oreochromis niloticus (n = 4).
* Site 4: X’pakay at 20 degrees 32 minutes 20.94 seconds North, 89 degrees 21 minutes 54.14 seconds West. Species: Rhamdia guatemalensis (n = 17) and Gambusia yucatana (n = 1).
* Site 5: Chonquilá at 20 degrees 38 minutes 15.94 seconds North, 89 degrees 20 minutes 39.44 seconds West. Species: Mayaheros urophthalmus (n = 2), Rhamdia guatemalensis (n = 13), and Gambusia yucatana (n = 2).
* Site 6: Chomin at 20 degrees 38 minutes 54.913 seconds North, 89 degrees 20 minutes 33.557 seconds West. Species: Rhamdia guatemalensis (n = 9) and Gambusia yucatana (n = 1).
* Site 7: Becal at 20 degrees 34 minutes 37.97 seconds North, 89 degrees 16 minutes 24.4 seconds West. Species: Rhamdia guatemalensis (n = 5) and Gambusia yucatana (n = 5).
* Site 8: Lum há at 20 degrees 36 minutes 8.03 seconds North, 89 degrees 15 minutes 36.65 seconds West. Species: Gambusia yucatana (n = 12).
* Site 9: X’kapnut at 20 degrees 37 minutes 23.376 seconds North, 89 degrees 6 minutes 35.143 seconds West. Species: Mayaheros urophthalmus (n = 4) and Rhamdia guatemalensis (n = 8).
* Site 10: Ixinha at 20 degrees 37 minutes 49.26 seconds North, 89 degrees 6 minutes 38.699 seconds West. Species: Rhamdia guatemalensis (n = 31).
* Site 11: Hotzó at 20 degrees 54 minutes 14.748 seconds North, 88 degrees 51 minutes 41.536 seconds West. Species: Mayaheros urophthalmus (n = 16).
* Site 12: X-colac at 20 degrees 55 minutes 19.058 seconds North, 88 degrees 52 minutes 24.275 seconds West. Species: Mayaheros urophthalmus (n = 10).
* Site 13: Chen Vazquez at 21 degrees 8 minutes 54.17 seconds North, 88 degrees 39 minutes 28.26 seconds West. Species: Mayaheros urophthalmus (n = 16) and Gambusia yucatana (n = 11).
* Site 14: Itzincab at 21 degrees 13 minutes 39.04 seconds North, 88 degrees 40 minutes 45.44 seconds West. Species: Mayaheros urophthalmus (n = 6) and Rhamdia guatemalensis (n = 3).
* Site 15: Sabakja at 20 degrees 34 minutes 49.76 seconds North, 89 degrees 35 minutes 17.52 seconds West. Species: Mayaheros urophthalmus (n = 10), Rhamdia guatemalensis (n = 1), and Gambusia yucatana (n = 3).
Host examination and collection of metacercariae
Fish were euthanised by spinal severance (pithing) following the guidelines accepted by the American Veterinary Medical Association (AVMA 2020). Prior to fish dissection, individuals were labelled, identified following Gallardo-Torres et al. (Reference Gallardo-Torres, Badillo-Alemán, Rivera Félix, Rubio Molina, Galindo De Santiago, Loera-Pérez, García-Galano and Chiappa-Carrara2014), and their total length (TL) was measured to the nearest 1 mm. Fish were dissected, and body surface, internal organs, gills, eyes, and brain were placed in Petri dishes with 0.65% saline solution and separately examined under a stereomicroscope. All detected metacercariae were isolated, counted, excysted although some were free or released from the cyst during the isolation, fixed in near-boiling saline solution, and stored in vials with 99% ethanol for morphological and molecular analyses.
Morphological study
Individuals of collected metacercariae from different hosts and habitats were photographed, either live or after staining with Mayers’ paracarmin or Harris’ haematoxylin, and mounted in Canada balsam, using Motic Images Plus 3.1 (Motic Instruments, Inc., Richmond, Canada). Specimens were first sorted by morphotypes representing either a genus, family, or an unknown taxon according to the main diagnostic characteristics. In some cases, specific features of metacercariae such as the number of rows of spines on the oral sucker and the number of spines per row, or the foliaceous body and a distinct lack of ventral sucker were used to confirm their identification following previously published studies; then, specimens were sequenced. Some representative metacercariae of diplostomids, clinostomids and heterophyids were thus deposited in the National Collection of Helminths (CNHE), Mexico City under the accession numbers 12692–12697.
Molecular identification of metacercariae
Specimens were individually placed in tubes and digested at 56°C overnight in a solution containing 10 mM Tris-HCl (pH 7.6), 20 mM NaCl, 100 mM EDTA (pH 8.0), 1% Sarkosyl, and 0.1 mg/mL Proteinase K. After digestion, DNA was extracted using DNAzol (Molecular Research Center, Cincinnati, OH, USA) following the manufacturer’s instructions. DNA samples were stored at −20°C until PCR amplifications were performed. The primers 391 (5′-AGCGGAGGAAAAGAAACTAA-3′) and 536 (5′-CAGCTATCCTGAGGGAAAC-3′) (García-Valera and Nadler Reference García-Varela and Nadler2005) were used to amplify the 28S ribosomal RNA gene. The partial cytochrome c oxidase subunit I (COI mtDNA) was amplified with four sets of primers to facilitate comparison with previously sequenced specimens of the families Clinostomidae Lühe, 1901, Heterophyidae Leiper, 1909, and Diplostomidae Poirier, 1886: the primers 527F (5′-ATTCG(R)TTAAAT(Y)TKTGTGA-3′) and 528R (5′-CCAAACYAACACMGACAT-3′) (Sereno-Uribe et al. Reference Sereno-Uribe, Pinacho-Pinacho, García-Varela and Pérez-Ponce de León2013); the primers JB3 (5′-TTTTTTGGGCATCCTGAGGTTTAT-3′) and JB4.5 (5′-TAAAGAAAGAACATAATGAAAATG-3′) (Bowles et al. Reference Bowles, Blair and McManus1995); Dice1F (5′-ATTAACCCTCACTAAATTWCNTTRGATCATAAG-3′) and Dice14R (5′ TAATACGACTCACTATACCHACMRTAAACATATGATG-3′) (Van Steenkiste et al. Reference Van Steenkiste, Locke, Castelin, Marcogliese and Abbott2015) and the primers Plat-diploCOX1F (5′-CGTTTRAATTATACGGATCC-3′) and Plat-diploCOX1R (5′-AGCATAGTAATMGCAGCAGC-3′) (Moszczynska et al. Reference Moszczynska, Locke, McLaughlin, Marcogliese and Crease2009). PCR products were verified on a 1% agarose TAE gel using 6X TriTrack DNA loading dye; positive amplifications were purified with ExoSAP-IT cleanup reagent (Thermo Fisher Scientific, Waltham, MA, USA) and sent to the Laboratorio Nacional de Biodiversidad (LaNaBio) at the Instituto de Biología, Universidad Nacional Autónoma de México for Sanger sequencing. Generated sequences were assembled using Geneious Prime Software v. 4.8.4 (Biomatters, Auckland, New Zealand). These sequences were deposited in GenBank under the accession numbers 28S: PZ764778–PZ764806, COI: PZ765725–PZ765748 (Table 2). Alignments per family and molecular marker of the sequences generated in the present study, together with relevant sequences from GenBank, were performed using MUSCLE (Edgar Reference Edgar2004) as implemented in MEGA 11.0 (Tamura et al. Reference Tamura, Stecher and Kumar2021). Four alignments were built to include novel sequences generated in this study, one for representatives of Heterophyidae (28S), two for Diplostomidae (28S and COI), and one for Clinostomidae (COI). Information on each taxon used for phylogenetic analyses, including host species, microhabitat, developmental stage, country of origin, and reference, is presented in Supplementary Tables S1–S3.
Summary data of trematode taxa, their microhabitat within host, fish hosts, and sequence identity of metacercariae sampled from cenotes within the RoC, Yucatán, Mexico
Table 2. Long description
The table consists of eight columns: Trematode taxa, Microhabitat, Fish host, Cenote Number, GenBank accession numbers, Gen, Matching sequence, and Percent Identity.
Key data rows include:
* Ascocotyle nana: Found in the mesentery of Mayaheros urophthalmus at Cenote 3; 28 S gene matches Ascocotyle species MF980609 at 98.5 percent.
* Austrodiplostomum compactum: Found in eyes of Gambusia yucatana and Mayaheros urophthalmus at Cenotes 12 and 13; 28 S and C O I genes match A. ostrowskiae and A. compactum at 99.8 to 100 percent.
* Clinostomum cichlidorum: Found in fins, gills, and eyes of Mayaheros urophthalmus at Cenotes 12, 13, and 14; matches C. cichlidorum and Clinostomum species at 99.5 to 100 percent.
* Clinostomum heluans: Found in fins of Mayaheros urophthalmus at Cenotes 12 and 13; matches C. poteae and C. heluans at 98.5 to 100 percent.
* Clinostomum chaacci: Found in gill cavity and muscle of Rhamdia guatemalensis at Cenotes 7, 9, and 10; matches C. chaacci at 100 percent.
* Diplostomidae gen. sp.: Found in mesentery of Thorichthys meeki and Mayaheros urophthalmus at Cenotes 2 and 3; matches Diplostomidae species at 97.9 percent.
* Posthodiplostomum aztlanensis: Found in mesentery of Gambusia yucatana at Cenote 13; matches P. aztlanensis at 100 percent.
* Posthodiplostomum macrocotyle: Found in brain of Mayaheros urophthalmus at Cenote 12; matches P. macrocotyle at 100 percent.
* Schwartzitrema sp.: Found in eyes, brain, and mesentery of Mayaheros urophthalmus at Cenotes 11 and 12; matches S. macrotesticulata at 89.2 to 98.2 percent.
* Uvulifer sp. 1 and sp. 2: Found in gills, fins, and skin of Mayaheros urophthalmus and Thorichthys meeki; matches Uvulifer species at 100 percent.
a The sequence appears in GenBank as Clinostomum sp., but it corresponds to C. chaaci after the study by Sereno-Uribe et al. (Reference Sereno-Uribe, García-Varela, Pinacho-Pinacho and Pérez-Ponce de León2018).
b The sequence appears in GenBank as Posthodiplostomum sp., later identified as P. aztlanensis by Gonzalez-García et al. (Reference González-García, López-Jiménez, Ortega-Olivares, Sereno-Uribe, Pérez-Ponce de León and García-Varela2024).
BLAST (Basic Local Alignment Search Tool) search was conducted at the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/) to assess the identity of sequenced specimens and to find relevant sequences available in GenBank for phylogenetic analyses. Arbitrarily, sequence identity values ≥99% for 28S and 95% for COI were considered appropriate to reach species-level designations. Maximum likelihood (ML) phylogenetic analyses were run with RAxML-HPC2 on ACCESS (8.2.12) (Stamatakis Reference Stamatakis2014), and 1000 bootstrap replicates were conducted using CIPRES Science Gateway v3.3 (Miller et al. Reference Miller, Pfeiffer and Schwartz2010). Phylogenetic trees were constructed with FigTree v.1.4.4 (Rambaut Reference Rambaut2018). Pairwise genetic divergence was determined using uncorrected distances (p-distances) calculated in MEGA 11.0 with default settings.
Results
Metacercarial infection
Fish trematode metacercariae were detected in 70 of the 236 fish examined (30%). A total of 2477 specimens representing 12 trematode taxa were found parasitising Gambusia yucatana Regan, 1914, Mayaheros urophthalmus (Günther 1862), Thorichthys meeki Brind, 1918 and Rhamdia guatemalensis (Günther 1864) (Table 2); however, no metacercariae were detected infecting Poecilia velifera (Regan 1914), Rocio octofasciata (Regan 1903), and the invasive Oreochromis niloticus (Linnaeus 1758), or fish samples from the cenotes Sabtún, X’pakay, Chonquilá, Chomin and Lum há (N° 1, 4, 5, 6, and 8 in Figure 1). The cichlid M. urophthalmus harboured the highest number of taxa, with nine, followed by another cichlid, T. meeki with two; while G. yucatana and R. guatemalensis were infected only with one trematode taxa each. Most of the infected fish (54%) had metacercariae of a single trematode taxon. Infections with two or more taxa were observed in one specimen of T. meeki and were common in M. urophthalmus, with one fish from cenote X-colac harbouring up to six trematode taxa at metacercarial stage. Specimens were initially identified to genus or family level in most cases based on a morphological assessment, and then some individuals of each morphotype were sequenced for one or two molecular markers (Figure 2).
Photomicrographs of whole stained mounted metacercariae specimens of (A) Clinostomum heluans, (B) C. cichlidorum, (C) C. chaacci, (D) Austrodiplostomum compactum, (E) Diplostomidae gen. sp., (F) Schwartzitrema sp., and (G) live metacercaria of excysted Uvulifer sp. 1. Scale bars: 200 μm.
Figure 2. Long description
A collection of seven panels arranged in two rows. Each panel contains a black scale bar representing 200 mu m.
Top row:
Panel A: Clinostomum heluans. An elongated, pink-stained specimen with a distinct oral sucker at the top and a long, straight body showing internal tubular structures.
Panel B: C. cichlidorum. A wider, pink-stained specimen with a prominent circular ventral sucker in the upper third of the body.
Panel C: C. chaacci. A specimen with dark, dense internal staining in the upper half and a lighter, translucent posterior region.
Panel D: Austrodiplostomum compactum. An oval-shaped, heavily pink-stained specimen with a visible internal organ structure in the lower third.
Bottom row:
Panel E: Diplostomidae gen. sp. A slender, elongated specimen with a granular internal texture and a small, darker stained region at the posterior end.
Panel F: Schwartzitrema sp. A wider, pear-shaped specimen with two distinct dark spots near the anterior end and a tapered posterior.
Panel G: Uvulifer sp. 1. A live, unstained specimen appearing grey and translucent, showing a constricted mid-section that gives it a figure-eight shape.
Molecular identification and phylogenetic relationships
BLAST searches and the position and interrelationships of sequenced specimens in phylogenetic analyses were useful for corroborating and, in some cases, for identifying trematode metacercariae. Twelve trematode taxa were recognised (Table 2); these were included in the families Heterophyidae (1 taxon), Diplostomidae (8 taxa), and Clinostomidae (3 taxa). Seven of them were identified to species level based on the sequence identity of the 28S rRNA with sequences deposited in GenBank. In the case of the heterophyid A. nana, the metacercariae were excysted, live-photographed, and one row with 16 spines was observed (Figure 3); it was identified as belonging to A. nana; then, specimens were fixed and sequenced for the 28S gene. The newly sequenced isolate nested in a clade with Ascocotyle sp. from cercariae collected from the snail Aroapyrgus alleei Morrison, 1946, from Laguna Escondida, Veracruz, Mexico (Figure 4), with very low divergence values (0.2–0.6%), showing conspecificity, i.e., specimens from Veracruz correspond with A. nana.
Photomicrograph of live specimens of the metacercaria of Ascocotyle nana from Mayaheros urophthalmus. (A) Cyst, (B) Excysted metacercaria, and (C) oral sucker showing one row of oral spines (1a–16a), and four basal spines (1b–4b).
Figure 3. Long description
A three-panel photomicrograph labeled A, B, and C.
* Panel A: A circular cyst containing a coiled metacercaria. The cyst wall is thin and translucent. A scale bar in the bottom right indicates 50 micrometers.
* Panel B: An elongated, excysted metacercaria with a visible oral sucker at the top anterior end and internal organs visible through the translucent body. A scale bar in the bottom right indicates 100 micrometers.
* Panel C: A high-magnification view of the oral sucker. It features a central slit-like opening surrounded by a ring of sixteen oral spines labeled 1a through 16a in a clockwise arrangement. On the left side of the sucker, four basal spines are labeled 1b, 2b, 3b, and 4b, arranged in a vertical row. A scale bar in the bottom right indicates 10 micrometers.
Phylogenetic interrelationships among Ascocotyle spp. based on maximum likelihood (ML) analysis of partial 28S rRNA gene sequences. Nodal support values lower than 80% are not shown. The new sequences generated in this study are indicated in bold.
Figure 4. Long description
A maximum likelihood phylogenetic tree with a scale bar of 0.02 at the bottom. The tree is rooted by Pygidiopsis macrostomum K T 8 7 7 4 0 9 at the bottom.
Moving from top to bottom, the terminal nodes are grouped as follows:
* Top Clade: Includes Ascocotyle sp. M Z 5 7 0 1 2 1, M Z 5 7 0 1 0 9, M Z 5 7 0 1 1 3, M Z 5 7 0 1 0 2, and M Z 5 7 0 1 1 8. This group is sister to a highlighted bold entry, Ascocotyle nana P Z 7 6 4 8 0 6. Below this is a sub-clade with 100 percent support containing Ascocotyle pindoramensis K J 0 9 4 5 6 1 and M F 9 8 0 6 0 9.
* Second Clade: Supported by a value of 98, containing a large group of Ascocotyle sp. including P P 8 6 2 7 6 3, P P 8 6 2 7 6 4, M Z 5 7 0 1 2 2, O L 9 6 0 0 4 9, M Z 5 7 0 1 2 9, M Z 5 7 0 1 2 6, and M Z 5 7 0 1 2 5. This clade also includes Ascocotyle trentinii M Z 6 5 4 8 8 0 and Ascocotyle diminuta O R 5 3 7 9 0 3.
* Third Clade: A pair with 100 percent support consisting of Ascocotyle longa M F 9 8 0 6 1 1 and M F 9 8 0 6 1 3.
* Fourth Clade: Supported by a value of 100, containing several sub-groups. One sub-group includes Ascocotyle sp. P P 9 1 9 6 4 9, O R 5 3 7 9 1 1, O R 5 3 7 9 1 0, and O R 5 3 7 9 0 5. Another sub-group contains Ascocotyle patagoniensis M K 3 5 9 0 8 2. A third sub-group includes Ascocotyle sp. M Z 5 7 0 1 1 9 and M Z 5 7 0 1 2 0. A fourth sub-group includes Ascocotyle sp. M Z 5 7 0 1 1 0, M Z 5 7 0 1 1 1, and M Z 5 7 0 1 2 8.
* Bottom Clades: Ascocotyle cameliae M K 3 5 9 0 8 0 and Ascocotyle sp. K U 5 5 9 5 6 1 branch off closer to the root.
Moreover, the identification of four species was based on the sequence identity values of one mitochondrial and one nuclear gene taking into account the highest value in both cases; that included the following species: Austrodiplostomum compactum (Lutz 1928) Dubois, 1970, Clinostomum cichlidorum Sereno-Uribe, García-Varela, Pinacho-Pinacho and Pérez-Ponce de León, 2018, Clinostomum heluans Braun, 1899, and Clinostomum chaacci Sereno-Uribe, López-Jiménez, Gonzalez-García, Ortega-Olivares and García-Varela, 2025 (Table 2). The phylogenetic position of these taxa (Figures 5–7) corresponds with that of previous studies. The COI phylogenetic tree of clinostomids (Figure 7) clearly show that three separate species occur in freshwater fish of cenotes in Yucatán because isolates nest in well supported monophyletic clades with isolates of these species.
Phylogenetic interrelationships among diplostomid taxa based on maximum likelihood (ML) analysis of partial 28S rRNA gene sequences. Nodal support values lower than 80% are not shown. The new sequences generated in this study are indicated in bold.
Figure 5. Long description
The phylogenetic tree is a rectangular cladogram with a scale bar of 0.07 at the bottom. The root on the left leads to several major clades.
At the top, a large clade includes Posthodiplostomum species, with several new sequences in bold such as P. aztlanensis P Z 7 6 4 7 9 6 and P. macrocotyle P Z 7 6 4 7 8 7 highlighted in grey boxes.
Below this is a clade for Uvulifer species, where Uvulifer sp. 1 and sp. 2 (sequences P Z 7 6 4 7 7 9 through P Z 7 6 4 8 0 2) are bolded and shaded in grey.
The middle section contains diverse genera including Crassiphiala, Cercocotyla, Prodiplostomum, and Neocrodicidicola. A specific group of Diplostomidae gen. sp. (P Z 7 6 4 8 0 5 and P Z 7 6 4 8 0 4) is bolded and shaded.
Further down, a distinct clade of Schwartzitrema species is shown, with ten new sequences (P Z 7 6 4 7 9 2 to P Z 7 6 4 7 9 0) in bold and shaded grey.
Near the bottom, a clade for Austrodiplostomum compactum contains five bolded sequences (P Z 7 6 4 7 9 5 to P Z 7 6 4 7 9 8) in a grey box.
The tree terminates at the bottom with Alaria and Suchocyathocotyle species as outgroups. Nodal support values are indicated at the branching points, ranging from 80 to 100.
The remaining taxa were not identified to species level and included five members of the Diplostomidae, i.e., Diplostomidae gen. sp., Posthodiplostomum sp., Schwartzitrema sp., Uvulifer sp. 1 and Uvulifer sp. 2 (Table 2). The phylogenetic position of all sampled taxa, including those not identified to species level, was assessed with ML analyses. The phylogenetic analysis of the 28S gene retrieved a tree (Figure 5), where Diplostomidae gen. sp. nested in a clade that includes Cystodiplostomum hollyi Dubois, 1936 as the sister taxa of several species and taxa of former proterodiplostomids, including species in the genera Proterodiplostomum and the recently described Pipatrema Tkach, Achatz and Melo, 2026; these relationships were supported by a very high nodal support value (Figure 5). In the case of Posthodiplostomum sp., and the two species of Uvulifer, BLAST analyses revealed that they did not match any identified species of these genera, but their phylogenetic position on the tree (Figure 5) clearly show they represent independent lineages. The isolates of Uvulifer sp. 1 were identical to the metacercaria of Uvulifer sp. (MK568575), from Herichthys labridens (Pellegrin, 1903) from Mexico, and the 28S sequences of Uvulifer sp. 2 and the isolate MF568570 from H. labridens were also identical. The phylogenetic trees from the analyses of the 28S and COI datasets of Diplostomidae (Figures 5–6) grouped some of the novel sequences generated in the present study as the sister taxa of Schwartzitrema macroholdfastus Achatz, Ho, Fecchio and Tkach, 2025 and S. macrotesticulata Achatz, Ho, Montes, Barneche, Fecchio and Tkach, 2025, with high nodal support values. Considering the divergence values between our isolates and the recently described Schwartzitrema species in the 28S gene (1.8–2.0%) and COI (9.4–10.5%), as well as the fact that our eight novel isolates nest as their sister taxa, they may represent a new species or a previously described species without available genetic data for their adult forms.
Phylogenetic interrelationships among diplostomid taxa based on maximum likelihood (ML) analysis of partial COI mtDNA gene sequences. Nodal support values lower than 80% are not shown. The new sequences generated in this study are indicated in bold.
Figure 6. Long description
A rectangular cladogram rooted by Australapatemon mclaughlini K Y 5 8 7 4 0 6 at the bottom. The tree branches into several major clades from top to bottom.
* The top clade includes Austrodiplostomum compactum M Z 3 2 3 2 4 6 and Austrodiplostomum compactum P Z 7 6 5 7 2 5 in bold, followed by Tylodelphys species.
* The second major clade contains Diplostomum lunaschiae and Diplostomum ardeae.
* The third clade features Hysteromorpha species including H. ostrowskiae and H. corti.
* The fourth clade consists of various Diplostomum species such as D. alascense and D. numericum.
* The fifth clade includes Posthodiplostomum and Crassiphiala bulboglossa.
* The sixth clade contains Bolbophorus species.
* The seventh and largest clade features a dense cluster of Schwartzitrema species. A bolded group includes Schwartzitrema sp. with accession numbers P Z 7 6 5 7 4 3, P Z 7 6 5 7 4 8, P Z 7 6 5 7 4 7, P Z 7 6 5 7 4 4, P Z 7 6 5 7 4 1, P Z 7 6 5 7 4 5, P Z 7 6 5 7 4 0, P Z 7 6 5 7 4 2, and P Z 7 6 5 7 4 6. Below these are Schwartzitrema macroholdfastus and Schwartzitrema macrotesticulata.
* The eighth clade includes Cardiocephaloides species.
* The ninth clade contains Ichthyocotylurus and Cotylurus strigeoides.
Nodal support values are indicated at branching points, ranging from 80 to 100. A scale bar at the bottom represents 0.07 substitutions per site.
Phylogenetic interrelationships among Clinostomum spp. based on maximum likelihood (ML) analysis of partial COI mtDNA gene sequences. Nodal support values lower than 80% are not shown. The new sequences generated in this study are indicated in bold.
Figure 7. Long description
A maximum likelihood phylogenetic tree with a scale bar of 0.06 at the bottom. The tree is rooted by Ithyoclinostomum yamagutii M N 6 9 6 1 6 3 at the bottom.
From top to bottom, the major clades are:
* Clinostomum cichlidorum: A large cluster including bolded sequences P Z 7 6 5 7 3 4, P Z 7 6 5 7 3 3, P Z 7 6 5 7 3 2, P Z 7 6 5 7 2 7, P Z 7 6 5 7 2 6, and P Z 7 6 5 7 3 1. It includes a node with 99 support and collapsed branches for C. tataxumui n=5 and C. arquus n=4.
* Clinostomum chaacci: A cluster including bolded sequences P Z 7 6 5 7 3 5, P Z 7 6 5 7 3 7, P Z 7 6 5 7 3 8, P Z 7 6 5 7 3 9, and P Z 7 6 5 7 3 6. A collapsed branch for C. caffarae n=4 is below this with 90 support.
* Clinostomum marginatum and dolichorchum: A mixed clade with support values of 100 and 92. It includes C. attenuatum n=2, C. poteae n=4, and C. album n=5.
* Clinostomum heluans: A cluster with bolded sequences P Z 7 6 5 7 2 9, P Z 7 6 5 7 3 0, and P Z 7 6 5 7 2 8, supported by a node of 96.
* Lower Clades: Includes C. sinensis n=4, C. complanatum n=3, C. chabaudi n=5, C. ukolii n=5, C. cutaneum n=2, C. phalacrocoracis n=5, and C. tilapiae n=3.
* Basal Clade: Clinostomum brieni M H 2 5 3 0 4 5 and a collapsed branch for C. brieni n=4 with 99 support.
Chord diagram showing the relationships between hosts and trematodes sampled in cenotes of the Yucatán Peninsula. Host families indicated in colour. Green, Poeciliidae; Red, Heptapteridae; Light blue, Eleotridae; Yellow, Cichlidae; Blue, Characidae.
Figure 8. Long description
A radial diagram where the top half of the circle represents Trematoda taxa labeled A 1 through A 30, and the bottom half represents fish host species.
* Trematoda Taxa (Top Arc): Labeled A 1 to A 30, grouped by families including Clinostomidae (A 1 to A 5), Cryptogonimidae (A 6 to A 9), Diplostomidae (A 10 to A 20), Echinostomatidae (A 21 to A 22), Heterophyidae (A 23 to A 29), and Opisthorchiidae (A 30).
* Fish Hosts (Bottom Arc): Color-coded by family. Green (Poeciliidae) includes Belonesox belizanus, Poecilia velifera, Poecilia latipunctata, Gambusia yucatana, and Poecilia petenensis. Red (Heptapteridae) includes Rhamdia guatemalensis. Light blue (Eleotridae) includes Gobiomorus dormitor. Yellow (Cichlidae) includes Parachromis friedrichstahlii, Vieja melanurus, Thorichthys meeki, Rocio octofasciata, Petenia splendida, Cincelichthys pearsei, and Mayaheros urophthalmus. Blue (Characidae) includes Astyanax aeneus.
* Connections: Colored ribbons flow across the center of the circle connecting specific parasites to their hosts. The width of the ribbons indicates the strength or frequency of the relationship. Notable dense connections include yellow ribbons from Cichlidae hosts to various Diplostomidae (A 10 to A 20) and blue ribbons from Astyanax aeneus to Heterophyidae (A 23 to A 29).
Discussion
Twelve trematode species were found infecting freshwater fish in cenotes as metacercariae in this study (Table 2). Previous records of metacercariae infecting fish from cenotes in the Yucatán Peninsula (Quintana Roo and Yucatán states) based on morphological grounds reported the occurrence of 18 species: Acanthostomum astorquii Watson, 1976, Acanthostomum minimum Stunkard, Reference Stunkard1938, Ascocotyle ampullacea Miller and Harkema, 1962, Ascocotyle diminuta Stunkard and Haviland, 1924, Ascocotyle felippei Travassos, 1929, Ascocotyle macrostoma (Robinson 1956) Sogandares-Bernal and Lumsden, 1963, Ascocotyle megalocephala Price, 1932, Ascocotyle nana Ransom, 1920, Ascocotyle nunezae Scholz, Vargas-Vázquez, Vidal-Martínez and Aguirre-Macedo, 1997, Austrodiplostomum compactum (Lutz 1928) Dubois, 1970, Cladocystis trifolium (Braun 1901) Poche, 1926, Clinostomatopsis intermedialis (Lamont 1920) Lunaschi and Drago, 2009, Crocodilicola pseudostoma (Willemoes-Suhm 1870) Poche, 1926, Echinochasmus macrocaudatus Ditrich, Scholz and Vargas-Vazquez, 1996, Echinochasmis leopoldinae Scholz, Ditrich and Vargas-Vazquez, 1996, Oligogonotylus manteri Watson, 1976, Pelaezia loossi (Viguera 1957) Salgado-Maldonado and Aguirre-Macedo, 1991, Posthodiplostomum minimum (MacCallum 1921) Dubois, 1936 (herein reported as Posthodiplostomum cf. minimum. Six additional taxa were recorded but only determined up to genus or family level, i.e., Apharyngostrigea sp., Clinostomun cf. complanatum, Echinostomatinae gen. sp., Heterophyidae gen. sp., Uvulifer sp., and Proterodiplostomidae gen. sp. 2 (Pearse Reference Pearse, Pearse, Creaser and Hall1936; Scholz et al. Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Franco1995b, Reference Scholz, Vargas-Vázquez, Aguirre-Macedo and Vidal-Martínez1997b, Reference Scholz, Aguirre-Macedo and Salgado-Maldonado2001; Stunkard Reference Stunkard1938). Twenty-one of these 24 trematode taxa were originally reported by Scholz et al. (Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Franco1995b) as metacercariae parasitising 14 fish species in 26 out of the 50 cenotes sampled across the Peninsula of Yucatán. These authors acknowledged that due to the high number of metacercariae sampled in some fishes and the time-consuming effort to isolate them, only a few larvae were evaluated. Further work on some of them, however, allowed their proper identification. For instance, Scholz et al. (Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Franco1995b) reported Echinochasmus sp. 1 from M. urophthalmus and Echinochasmus sp. 2 from Astyanax aeneus. These taxa were later identified as Echinochasmus macrocaudatus (Ditrich et al. Reference Ditrich, Scholz and Vargas-Vázquez1996) and E. leopoldinae (Scholz et al. Reference Scholz, Ditrich and Vargas-Vázquez1996b), respectively; likewise, Proterodiplostomidae gen. sp. 1 from R. guatamalensis was considered conspecific with some specimens found in Hondo River in Quintana Roo identified as Crocodilicola pseudostoma (Scholz and Vargas-Vazquez Reference Scholz and Vargas-Vázquez1998), and Posthodiplostomum sp. actually corresponded with Uvulifer sp. (see Vidal-Martínez et al. Reference Vidal-Martínez, Aguirre-Macedo, Scholz, González-Solís and Mendoza-Franco2001). The same was true for several species of the heterophyid genus Ascocotyle identified by Scholz et al. (Reference Scholz, Vargas-Vázquez, Vidal-Martínez and Aguirre-Macedo1997a) as Ascocotyle nunezae, A. nana and A. megalocephala, previously designated as Ascocotyle (Phagicola) sp. 3, A. (Phagicola) sp. 2, and A. (Ascocotyle) sp. 1, respectively, by Scholz et al. (Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Franco1995b), while A. ampullacea was found after their manuscript was accepted (Scholz et al. Reference Scholz, Vargas-Vázquez, Aguirre-Macedo and Vidal-Martínez1997b).
Despite sampling five localities in common with Scholz et al. (Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Franco1995a, Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Francob), only two species were shared in both datasets, i.e., Austrodiplostomum compactum and Ascocotyle nana. The overall difference in helminth species richness between the studies by Scholz and co-workers in the decade of the 1990s and the present study might be certainly related to the unequal sampling effort. For instance, previous studies covered a larger geographical area across the Yucatán Peninsula, not just the Yucatán state. Additionally, the number of fish examined (581 vs. 236 fish), host species examined (15 vs. 7), and number of cenotes sampled (39 vs. 15) were lower in the present study. Irrespective of these differences, the metacercarial composition between the past and the present study is basically related to the number of species of Ascocotyle and a few more that were not sampled in this study. Indeed, Ascocotyle a hitherto considered dominant component of the helminth fauna of cenote fish, was represented only by A. nana in the present study; this probably reflects the variation in the faunal composition of coastal and inland cenotes, and consequently the occurrence of suitable hosts; for instance, the definitive hosts of Ascocotyle spp. are fish-eating birds, which are usually more abundant in cenotes closer to the line coast (Scholz et al. Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Franco1995b), where some other fish species are found such as Astyanax aeneus (Günther 1860), Petenia splendida Günther, 1862, Poecilia petenensis Günther, 1866, and Belonesox belizanus Kner, 1860 that serve as their second intermediate host and were not found in cenotes within the RoC, this could also explain the absence of metacercariae of Echinochasmus spp. and Apharyngostrigea sp. in this study.
However, and even though it might seem premature to hypothesise, overall results suggest an emerging pattern of change pointing towards a reduction in the diversity of trematode metacercariae parasitising fish from cenotes in the last 30 years that requires more detailed evaluation and an increase in the number of sampled cenotes across a wider geographical range and host individuals. In spite of the higher level of precision in the identification of metacercariae, species diversity was lower than that described in the decade of the 1990s by Scholz and co-workers. This reduction is probably related to alterations in the trophic status and water quality of cenotes due to a rapid demographic and economic expansion that has been taking place in the last decades, in addition to the lack of wastewater treatment or sewage systems (Arcega-Cabrera et al. Reference Arcega-Cabrera, Velázquez-Tavera, Fagher, Derrien and Noreña-Barroso2014), but also due to contamination with banned organochlorine pesticides and organic pollutants (Moreno-Pérez et al. Reference Moreno-Pérez, Hernández-Téllez and Bautista-Galvez2021; Polanco-Rodríguez et al. Reference Polanco-Rodríguez, Navarro-Alberto, Solorio-Sánchez, Mena-Rejón, Marrufo-Gómez and Del Valls-Casillas2015). Another interesting trend observed in the present study was the host specificity of trematode metacercariae towards their hosts, either at species or family level (Figure 8). Patterns of host specificity were unravelled through a more precise species delimitation using molecular data. For example, novel data allowed the identification of three congeneric species of the genus Clinostomum infecting cenote-dwelling fishes as metacercariae (Table 2), although only one species (Clinostomum cf. complanatum) was previously reported infecting A. aeneus, R. guatemalensis and M. urophthalmus (Scholz et al. Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Franco1995b). In the present study, C. chaacci was only found infecting the heptapterid R. guatemalensis, whereas the cichlid M. urophthalmus harboured metacercariae of two species characteristic of Middle American cichlids, C. cichlidorum and C. heluans (Figure 8) (Briosio-Aguilar et al. Reference Briosio-Aguilar, Pinto, Rodríguez-Santiago, Lopez-García, García-Varela and Pérez-Ponce de León2018; Pérez-Ponce de León et al. Reference Pérez-Ponce de León, García-Varela, Pinacho-Pinacho, Sereno-Uribe and Poulin2016; Sereno-Uribe et al. Reference Sereno-Uribe, García-Varela, Pinacho-Pinacho and Pérez-Ponce de León2018, Reference Sereno-Uribe, López-Jiménez, González-García, Ortega-Olivares and García-Varela2025). The finding of C. heluans represents the first report of the metacercaria of the species in Mexico, contributing to elucidate the life cycle of C. heluans in its northernmost distribution range, and a new host record that further confirms the affinity for cichlids as second intermediate hosts of these metacercariae, which have been previously found in Cichlasoma boliviense Kullander, 1983 in Bolivia, Cichlasoma dimerus (Heckel 1840) in Argentina, Cichla temensis Humboldt, 1821, Geophagus proximus (Castelnau 1855) and Australoheros sp. in Brazil (Briosio-Aguilar et al. Reference Briosio-Aguilar, Pinto, Rodríguez-Santiago, Lopez-García, García-Varela and Pérez-Ponce de León2018; Locke et al. Reference Locke, Caffara, Marcogliese and Fioravanti2015; Montes et al. Reference Montes, Barneche, Pagano, Ferrari, Martorelli and Pérez Ponce de León2021; Vicente et al. Reference Vicente, Santos and Souza1978; Zago et al. Reference Zago, Franceschini, Zocoller-Seno, Veríssimo-Silveira, Maia, Ikefuti and Silva2012).
The approach followed in the present study, incorporating molecular data, did not reveal a higher diversity of trematode metacercaria in freshwater fish from cenotes, in contrast to many recent studies applying molecular identification (e.g., Kudlai et al. Reference Kudlai, Binkienė, Rakauskas and Baker2026; Truter et al. Reference Truter, Yong, Smit, Chakona, Luus-Powell and Smit2025). Nevertheless, it was very important to refine certain species hypotheses. For example, in this study, three species of Posthodiplostomum, i.e., P. aztlanensis ex G. yucatana, P. macrocotyle ex M. urophthalmus, and Posthodiplostomum sp. ex M. urophthalmus, were found. In the previous study by Scholz and co-workers, only P. minimum was reported in cichlids, as well as an undetermined form of Posthodiplostomum which turned out to correspond to Uvulifer (Vidal-Martínez et al. Reference Vidal-Martínez, Aguirre-Macedo, Scholz, González-Solís and Mendoza-Franco2001). The case of P. minimum is particularly interesting. Scholz et al. (Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Franco1995b) reported P. minimum as a parasite of three species of cichlids in cenotes of Yucatán. Actually, P. minimum was the only congeneric species occurring in freshwater fish of Mexico, with an allegedly wide distribution across Mexico and Central America, infecting a wide array of freshwater fish (see Pérez-Ponce de León et al. Reference Pérez-Ponce de León, García-Prieto and Mendoza-Garfias2007, Reference Pérez-Ponce de León, Sereno-Uribe, Pinacho-Pinacho and García-Varela2022). Based on the premise that this species was the only one in that geographical area, Pérez-Ponce de León et al. (Reference Pérez-Ponce de León, Sereno-Uribe, Pinacho-Pinacho and García-Varela2022) sequenced specimens of metacercariae from different host species and localities and discovered six putative species, although none of them was assigned to P. minimum. At that time, the only available sequences of P. minimum were those from the study of Kvach et al. (Reference Kvach, Jurajda, Bryjová, Trichkova, Ribeiro, Přikrylová and Ondračková2017) (identified as Posthodiplostomum cf. minimum) from centrarchids in eastern Europe (GenBank MF171004, MF170998), although it was later demonstrated through the ITS divergence that this species corresponded to P. centrarchi. Still, none of the lineages uncovered by Pérez-Ponce de León et al. (Reference Pérez-Ponce de León, Sereno-Uribe, Pinacho-Pinacho and García-Varela2022) nested with these sequences. Unfortunately, that study did not include sequence data of specimens from the Yucatán Peninsula. For convenience, we suggest that the record of P. minimum of Scholz et al. (Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Franco1995b) should be considered as Posthodiplostomum cf. minimum, requiring further verification. However, the present data confirmed that at least three species of Posthodiplostomum occur in fish from cenotes: Posthodiplostomum sp., P. macrocotyle, and P. aztlanensis. The latter species was described from ardeid birds from Neotropical areas of Mexico and Central America (González-García et al. Reference González-García, López-Jiménez, Ortega-Olivares, Sereno-Uribe, Pérez-Ponce de León and García-Varela2024), and adults were linked to metacercariae Clade V of the study by Pérez-Ponce de León et al. (Reference Pérez-Ponce de León, Sereno-Uribe, Pinacho-Pinacho and García-Varela2022). In a recently published study, Burris-Otero et al. (Reference Burris-Otero, Achatz, Marcogliese, Reyda, Colston and Locke2026) suggested the possibility that P. aztlanensis and three other species found in North America (P. dawnsherryae, P. nanum, and P. seminolense) might be junior synonyms of P. minimum based on morphology. However, this hypothesis needs to be tested since, as the authors discussed, no sequence data of the type host and locality of P. minimum are currently available, and also, it has to be considered that Achatz et al. (Reference Achatz, Von Holten, Pritchard and Keel2025) uncovered a high genetic divergence (0.5–5.9% in partial 28S sequences and 10.9–17.4% in partial cox1 sequences) among the four species that Burris-Otero et al. (Reference Burris-Otero, Achatz, Marcogliese, Reyda, Colston and Locke2026) suggested as potential synonyms of P. minimum.
Regarding the metacercariae of the former family Proterodiplostomidae (now considered Diplostomatidae, see Achatz and Tkach Reference Achatz and Tkach2025) reported by Scholz et al. (Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Franco1995b), considering the lack of genetic data from these specimens, it would be impossible to determine their relationship to the metacercariae of Diplostomidae gen. sp. found during this study, whose identity remains unclear. In Mexico, Crocodilicola pseudostoma is the only species related to previously considered proterodiplostomids reported in fish; it has been found in diverse host species such as Astyanax aeneus, Dormitator maculatus (Bloch 1792), Poecilia mexicana Steindachner, 1863, Rhamdia guatemalensis, and the cichlids M. urophthalmus and Thorichthys aureus (Günther 1862) (see references in Pérez-Ponce de León et al. Reference Pérez-Ponce de León, García-Prieto and Mendoza-Garfias2007 and García-Prieto et al. Reference García-Prieto, Dáttilo, Rubio-Godoy and Pérez-Ponce de León2022). The phylogenetic analysis suggested no close relationship of our isolates to the only sequenced specimens designated as C. pseudostoma (MF398328) by Hernández-Mena et al. (Reference Hernández-Mena, García-Varela and Pérez-Ponce de León2017). Taking into account that reports of adults and metacercariae of C. pseudostoma in Mexico have been considered questionable (see Moravec Reference Moravec2001, Tkach et al. Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and de Vasconcelos Melo2020) and that the identity of the metacercariae sampled in the present study, as those reported as Proterodiplostomidae gen. sp. 2 by Scholz et al. (Reference Scholz, Vargas-Vázquez, Moravec, Vivas-Rodríguez and Mendoza-Franco1995b) remains uncertain, the actual diversity of trematodes considered as members of the former family Proterodiplostomatidae in the region can only be unravelled by sequencing adults that could be linked to larval stages, and by adding more related species to the genetic library.
This study significantly increased the genetic library of metacercariae parasitising cenote-dwelling fish, apart from some few exceptions already mentioned. Only seven of the 24 taxa previously reported in cenotes have sequences available in GenBank from other localities of at least one mtDNA or rDNA gen/region, since their distribution ranges extend beyond the Yucatán Peninsula. Future assessments of trematode species diversity and other organisms in vulnerable ecosystems such as cenotes could benefit from strengthening a more comprehensive genetic library, since it would allow the application of novel and less invasive methodology such as environmental DNA-based methods, that could avoid the impact of sampling fish in anthropogenically impacted water bodies such as cenotes, facilitate the intrinsic challenges of sampling in these water bodies, and ultimately the use of these methods could result in more accurate estimations of species diversity and a better understanding of parasite life cycles and host-parasite relationships.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X2610234X.
Data availability
DNA sequence data were deposited in GenBank under the accession numbers PZ764778–PZ764806, PZ779948, and PZ765725–PZ765748.
Acknowledgements
This paper represents one of the fulfillments for CMM to complete the PhD program of the Posgrado en Ciencias del Mar y Limnología (field of knowledge: Limnology), National Autonomous University of Mexico (UNAM). CMM wishes to thank the Posgrado en Ciencias del Mar y Limnología of the National Autonomous University of Mexico for her studies within this program to obtain her PhD degree and SECIHTI for the scholarship to complete this PhD program (CVU 1249633). LAG thanks SECIHTI for the postdoctoral fellowship ‘Estancias Posdoctorales por México Modalidad Académica’ (CVU 640068). We greatly appreciate the support of Laura Marquez and Nelly López, LaNaBio, for sequencing DNA. We thank Norberto Colín for allowing us to use the Molecular Biology laboratory of ENES-Mérida, Maribel Badillo and Alfredo Gallardo for the confirmation of the fish species identification, and Francisco Puc Itza and Oscar A. Centeno Chale for their help during field work.
Financial support
This study was funded by a grant from the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica PAPIIT-UNAM IN 200824 to GPPL.
Competing interests
The authors declare no competing interests.
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