Introduction
Adults of Neodiplostomum Railliet, 1919, are distributed worldwide as parasites, mainly of the intestines of birds of prey, and occasionally those of passerines, cuculiforms, and mammals (Achatz et al Reference Achatz, Chermak, Martens, Pulis, Fecchio, Bell, Greiman, Cromwell, Brant, Kent and Tkach2021, Reference Achatz, Kostadinova, Georgieva, Fecchio and Tkach2025; Cacique et al Reference Cacique, Barriga, Bello, Pino, Salcedo-Mejia, Ñacari, Crucces, Mondragón-Martínez and Chero2025; Dubois Reference Dubois1970; Niewiadomska Reference Niewiadomska, Gibson, Jones and Bray2002). Freshwater snails from the families Planorbidae, Physidae, and Lymnaeidae serve as the first intermediate hosts (Blasco-Costa and Locke Reference Blasco-Costa, Locke, Rollinson and Stothard2017), whereas metacercariae have been recorded in amphibians, which serve as second intermediate hosts (Blasco-Costa and Locke Reference Blasco-Costa, Locke, Rollinson and Stothard2017; Chandler Reference Chandler1942; Izrailskaia and Besprozvannykh Reference Izrailskaia and Besprozvannykh2023). Recently, species of Neodiplostomum have been targeted for numerous morphological and molecular studies, and their taxonomy and systematics have started to be resolved (see Achatz et al Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022, Reference Achatz, Kostadinova, Georgieva, Fecchio and Tkach2025; Cacique et al Reference Cacique, Barriga, Bello, Pino, Salcedo-Mejia, Ñacari, Crucces, Mondragón-Martínez and Chero2025; Heneberg et al Reference Heneberg, Sitko and Těšínský2020; Woodyard et al Reference Woodyard, Rosser and Griffin2017). A taxonomic review of Neodiplostomum by Woodyard et al (Reference Woodyard, Rosser and Griffin2017), Achatz et al (2022, Reference Achatz, Kostadinova, Georgieva, Fecchio and Tkach2025), and Martinez et al (Reference Martinez, Flores, Rauque and Viozzi2026) revealed at least 77 described species worldwide: 15 in North America, 13 in South America, and a single species in Middle America, representing 37.6% of the diversity of Neodiplostomum in the Americas (see Table 1).
List of species of Neodiplostomum from the Americas
Table 1. Long description
The table consists of two columns: Taxon name and Distribution.
North America Distribution:
* N. accipitris Dubois and Rausch, 1948
* N. americanum Chandler and Rausch, 1947
* N. attenuatum Dubois and Macko, 1972
* N. banghami Penrod, 1947
* N. centuri Dubois and Macko, 1972
* N. caballeroi Zerecero, 1943
* N. buteonis Dubois and Rausch, 1948
* N. delicatum Chandler and Rausch, 1947
* N. cratera Barker and Noll, 1915
* N. lucidum La Rue and Bosma, 1927
* N. isomegalocotyle Dubois and Macko, 1972
* N. pearsoni Dubois, 1962
* N. reflexum Chandler and Rausch, 1947
* N. laurei Miller, 1940
* N. paraspathula Noble, 1936
South America Distribution:
* N. ellipticum Brandes, 1888
* N. fastigatum Lutz, 1928
* N. pitangi Dubois, 1985
* N. nephrocystis Lutz, 1928
* N. cornutum Achatz, Kostadinova, Georgieva, Fecchio and Tkach, 2025
* N. lutzi Achatz, Kostadinova, Georgieva, Fecchio and Tkach, 2025
* N. branchiocystis Lutz, 1928
* N. conicum Dubois, 1937
* N. vaucheri Dubois, 1983
* N. microcotyle Dubois, 1937
* N. travassosi Dubois, 1937
* N. sparverius Martinez, Flores, Rauque, Viozzi, 2026
* N. caracara Martinez, Flores, Rauque, Viozzi, 2026
Central America Distribution:
* N. morenodathei Odening, 1970
The first record of a Neodiplostomum species in Mexican territory dates back to 1943, when Zerecero described the species N. caballeroi from the brown rat, Rattus norvegicus Berkenhout. Later, Caballero (Reference Caballero1944) recorded the species N. paraspathula from the intestine of a golden eagle, Aquila chrysaetos L., from San Luis Potosi. Finally, a third species, N. cratera, was recorded from the raccoon, Procyon lotor L. However, this record could not be verified because the sample was not deposited in any helminthological collection (see Pérez-Ponce de León et al Reference Pérez-Ponce de León, García-Prieto and Mendoza-Gárfias2007). In the last taxonomic review of Neodiplostomum from South America, Achatz et al (Reference Achatz, Kostadinova, Georgieva, Fecchio and Tkach2025) reported that many diplostomid species still await discovery because of the high biodiversity and potential definitive hosts that inhabit the Neotropical region. During several helminthological surveys in the Neotropical region of Mexico, specimens belonging to the genus Neodiplostomum were identified, revealing a combination of unique morphological traits. The main objectives of the current study were (i) to describe three new species of Neodiplostomum obtained from five definitive hosts; (ii) to molecularly characterise the three new species of Neodiplostomum by using four molecular markers (three nuclear and one mitochondrial DNA marker); (iii) to test the phylogenetic position of these three species within the genus Neodiplostomum; and (iv) to explore the ecological associations of the three new species of Neodiplostomum with their definitive hosts.
Materials and methods
Specimen collection and morphological analyses
A total of 34 birds were collected between December 2019 and December 2025 in Tlacotalpan, Veracruz (18° 37′ 24.8″ N 95° 39′ 17.24″ W) from the Neotropical region of Mexico (Table 2). Two individuals of roadside hawk, Rupornis magnirostris Gmelin, 17 groove-billed ani, Crotophaga sulcirostris Swainson, 6 common squirrel-cuckoo, Piaya cayana L, 8 great kiskadee, Pitangus sulphuratus L, and 1 boat-billed flycatcher, Megarynchus pitangua L, representing three families, Accipitridae, Cuculidae, and Tyrannidae. The birds were identified based on morphological characteristics following Howell and Webb (Reference Howell and Webb1995), Van Perlo (Reference Van Perlo2006), and the American Ornithologists’ Union (1998), until the 65th update (Chesser et al. Reference Chesser, Billerman, Burns, Cicero, Dunn, Hernández-Baños, Jiménez, Johnson, Kratter, Mason, Rasmussen and Remsen2024). Adult digeneans were removed from the intestine of five bird species, placed in Petri dishes with 0.6% saline solution, and examined under a stereomicroscope (EZ4 Leica). Adult diplostomids were relaxed with heat-distilled water and preserved in 70% ethanol for DNA analyses. Additionally, other specimens were fixed in hot 4% formalin for scanning electron microscopy studies. Specimens preserved in 70% ethanol were stained with Mayer’s paracarmine (Merck, Darmstadt, Germany), dehydrated in a graded ethanol series, cleared with methyl salicylate, and mounted on permanent slides with Canada balsam. Specimens were photographed and measured using a Leica DM 1000 LED compound microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany); measurements are reported in micrometres (μm). Internal morphological features were illustrated using a drawing tube attached to a Leica MC120HD microscope. Drawings were made using Adobe Illustrator 27.9 (Adobe, Inc., San Jose, CA, USA). Types and paratypes were deposited in the Colección Nacional de Helmintos (CNHE), Instituto de Biología, Universidad Nacional Autónoma de México (UNAM), Mexico City.
GenBank accession numbers, host, country, species name of Neodiplostomum spp., used in this study
Table 2. Long description
The table contains 9 columns: Species, Host species, Family species, Country, S S U, L S U, I T S, Cox 1, and Source. Key entries include:
* Neodiplostomum acatlotlii n. sp. from Rupornis magnirostris in Mexico, with L S U P Z 535817–820 and Cox 1 P Z 535945–948.
* Neodiplostomum papaloapanensis n. sp. from Crotophaga sulcirostris and Piaya cayana in Mexico, with Cox 1 P Z 535949–950 and P Z 537042.
* Neodiplostomum leoni n. sp. from Pitangus sulphuratus and Megarynchus pitangua in Mexico, with Cox 1 P Z 535951–952 and P Z 537043.
* Neodiplostomum americanum found in various hosts in the U S A including Bubo virginianus, Megascops asio, and Lithobates pipiens, with I T S markers like K Y 851307–08 and Cox 1 O L 770047–48.
* Neodiplostomum banghami from Thamnophis sirtalis and Falco columbarius in the U S A.
* Neodiplostomum vaucheri from Trachops cirrhosus in Ecuador.
* Neodiplostomum cf. cratera (types 1, 2, and 3) from various North American mammals and amphibians like Taxidea taxus and Procyon lotor.
* Neodiplostomum seoulense from Rattus norvegicus in Russia.
* Neodiplostomum reflexum from Buteo jamaicensis and Strix varia in the U S A.
* Neodiplostomum microcotyle from Busarellus nigricollis in Brazil.
* Neodiplostomum attenuatum and Neodiplostomum sphatula from the Czech Republic.
* Neodiplostomum cornutum, N. nephrocystis, N. fastigatum, N. lutzi, and N. ellipticum from various hosts in Brazil, with P P prefix accession numbers.
Additionally, some specimens preserved in 4% formalin were dehydrated in a graded ethanol series, critical point dried with CO2, sputter-coated with gold, and examined with a Hitachi Stereoscan Model S-2469N scanning electron microscope at 15 kV at LaNABIO, Instituto de Biología, Universidad Nacional Autónoma de México (UNAM).
Molecular study
Prior to the extraction of the genomic DNA, specimens preserved in ethanol were mounted on a microscope slide, and images were taken as references with a bright-field Leica DM 1000 LED microscope (Leica, Wetzlar, Germany). Each image was linked with its genomic DNA (photogenophore sensu Andrade-Gómez and García-Varela Reference Andrade-Gómez and García-Varela2021) (Figure 1). Each specimen was removed from the microscope slide and genomic DNA was isolated, following the protocol described by González-García et al. (Reference González-García, Ortega-Olivares, Andrade-Gómez and García-Varela2020). Three regions of nuclear ribosomal DNA (rDNA) were amplified using the polymerase chain reaction (PCR). Near-complete small subunit (SSU) (~1,800 bp) was amplified using two overlapping PCR fragments of 1,000 bp. The primers used for SSU amplicon one were forward 18S1A, 5′-GGCGATCGAAAAGATTAAGCCATGCA-3′ and reverse 32, 5′-CGAAGTCCTATTCCATTATTC-3′; for amplicon two, forward 652, 5′-GCAGCCGCGGTAATTCCAGCTC-3′ and reverse 28, 5′-AGCGACGGGCGGTGTGT-3′. The domains D1–D3 from the large subunit (LSU) (~1,200 bp) were amplified using the forward primer 391, 5′-AGCGGAGGAAAAGAAACTAA-3′ (Nadler et al Reference Nadler, Hoberg, Hudspeth and Rickard2000), and the reverse primer 536, 5′-CAGCTATCCTGAGGGAAAC-3′ (García-Varela and Nadler Reference García-Varela and Nadler2005). The ITS region, including ITS1, 5.8S, and ITS2 (ITS), was amplified using the forward primer BD1 5′-GTCGTAACAAGGTTTCCGTA-3′ (Bowles and McManus Reference Bowles and McManus1993) and the reverse primer BD2 5′-ATCTAGACCGGACTAGGCTGTG-3′ (Bowles et al Reference Bowles, Blair and McManus1995). The cox 1 gene was amplified using the forward primer PosthoMex 5′-GTCCHGAGGTTTATAAWTATGT-3′ and the reverse primer JB4, 5′-TAAAGAACATAATGAAATTG-3′ (Bowles et al Reference Bowles, Blair and McManus1992). The amplification reactions were performed with MyTaq DNA polymerase (Bioline Laboratories, London, UK) following the manufacturer’s instructions in a total volume of 12.5 μL of each PCR reaction, containing 1 μL of each primer, 10 pmol/μL, plus 2.0 μL of genomic DNA. PCR cycling parameters for DNA amplification included denaturation at 94 °C for 3 min, followed by 35 cycles of 94 °C for 1 min, annealing at 50°C (SSU, LSU, ITS) and 40 °C (cox 1) for 1 min, and extension at 72 °C for 1 min, followed by postamplification incubation at 72 °C for 7 min. Sequencing reactions were performed with the primers mentioned above using ABI Big Dye (Applied Biosystems, Boston, Massachusetts) terminator sequencing chemistry. The reaction products were separated and detected using an ABI 3730 capillary DNA sequencer. The contigs were assembled, and base-calling differences were resolved using Codoncode Aligner version 12.0.1 (Codoncode Corporation, Dedham, Massachusetts).
Photogenophores of Neodiplostomum acatlotlii n. sp., from Rupornis magnirostris (A); Neodiplostomum papaloapanensis n. sp., from Crotophaga sulcirostris (B); Neodiplostomum leoni n. sp., from Pitangus sulphuratus (C).
Alignments and phylogenetic analyses
Newly generated sequences of SSU, LSU, ITS, and cox 1 were aligned with other diplostomid sequences available in GenBank (Table 2). Sequences of each molecular marker were aligned using SeaView v.4 (Gouy et al Reference Gouy, Guindon and Gascuel2010) and adjusted with the Mesquite program (Maddison and Maddison Reference Maddison and Maddison2011). The nucleotide substitution model was selected using jModelTest v.2.1.7 (Darriba et al Reference Darriba, Taboada, Doallo and Posada2012) applying the Akaike information criterion. The best nucleotide substitution model was selected for each dataset. Phylogenetic analyses were reconstructed through Bayesian inference (BI) and Maximum likelihood (ML) using the online interface Cyberinfrastructure for Phylogenetic Research (CIPRES) Science Gateway v3.3 (Miller et al. Reference Miller, Pfeiffer and Schwartz2010). BI analysis was inferred with MrBayes v.3.2.7 (Ronquist et al Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012), with two simultaneous runs of the Markov Chain Monte Carlo (MCMC) for 10 million generations, sampled every 1000 generations, using a heating parameter value of 0.2 and a burn-in of 25%. ML analysis was carried out with RAxML v.7.0.4 (Silvestro and Michalak Reference Silvestro and Michalak2011), and 1000 bootstrap replicates were run to assess nodal support. Phylogenetic trees were drawn and edited in FigTree v.1.3.1 (Rambaut Reference Rambaut2012). Genetic divergence among taxa was estimated using uncorrected ‘p’ distances with MEGA6 (Tamura et al Reference Tamura, Stecher, Peterson, Filipski and Kumar2013).
Results
Morphological description
Diplostomidae Poirier, 1886
Neodiplostomum Railliet, 1919
Neodiplostomum acatlotlii n. sp.
Type-host: Rupornis magnirostris Gmelin, 1788.
Site of infection: Intestine
Type locality: Tlacotalpan, Veracruz, Mexico (18° 37′ 24.8″ N 95° 39′ 17.24″ W).
Type material: CNHE: 12590 (holotype); 12591 (paratypes).
Representative DNA sequences: PZ535833 (SSU); PZ535817-820 (LSU); PZ535825-828 (ITS); PZ535945-948 (cox 1).
Etymology: The specific epithet refers to acatlotli, which means hawk in the Náhuatl language; the Nahuas are an ancient group that descended from the Aztecs, who inhabited central and northern Mexico.
Description (based on 13 adult specimens plus two specimens for SEM); measurements of holotype (Figures 2A and 3A–D) given in text; measurements of the entire series given in Table 3. Body distinctly bipartite, cylindrical, with a rounded posterior end, 1.4 mm long. Prosoma cylindrical, concave, 949 long, covered with tiny spines, widest at the level of the holdfast organ 308. Opisthosoma oval, unarmed, 480 long, narrower than prosoma, 283 wide. Prosoma-opisthosoma length ratio 1.9. Oral sucker terminal muscular, subspherical, 36 × 45, surrounding with 7 small dome-like papillae (Figure 3B). Ventral sucker muscular, subspherical, surrounding with two rings of small dome-like papillae, larger than the oral sucker, 50 × 45, at mid-length of prosoma (Figure 3C). Oral sucker: ventral sucker width ratio 0.98. Holdfast organ oval in the last quarter of the prosoma with minute spines, 202 × 145. Proteolytic gland dorsal to the posterior part of the holdfast organ. Prepharynx absent. Pharynx muscular, oval, 39 × 32. Oesophagus absent. Caeca bifurcation in the most anterior quarter of the prosoma immediately to the pharynx. Caeca slender, reaching the posterior end of the body. Testes two, tandem, contiguously separated. Anterior testis asymmetrical 104 × 194, posterior testis somewhat bilobed 110 × 209. Seminal vesicle not observed. Ovary elliptical, pretesticular, placed between the anterior testis and the prosoma-opisthosoma junction 66 × 102. Vitellarium located from near the caecal bifurcation in the prosoma, extending to the opisthosoma to the posterior end of the body. Egg, 48 × 31. Excretory pore terminal.
Drawings of Neodiplostomum acatlotlii n. sp., from Rupornis magnirostris (A); Neodiplostomum papaloapanensis n. sp., from Crotophaga sulcirostris (B); Neodiplostomum leoni n. sp., from Pitangus sulphuratus (C); whole worm, holotype, ventral view.
Figure 2. Long description
A three-panel scientific illustration showing the ventral anatomy of three parasitic flatworms.
Panel A, on the left, depicts Neodiplostomum acatlotlii. It has an elongated, slender body with a slight constriction in the middle. At the anterior end is a small oral sucker, followed by a pharynx and a ventral sucker located in the upper third of the body. Below the ventral sucker is a large, lobed tribocytic organ. The posterior half contains two large, tandem testes and a smaller ovary. A scale bar to the left indicates 500 mu m.
Panel B, in the center, depicts Neodiplostomum papaloapanensis. This species has a much broader, more oval-shaped anterior segment and a distinct constriction separating it from the posterior segment. The tribocytic organ is large and centrally located in the anterior segment. The posterior segment is filled with large, granular testes and a vertical row of eggs. A scale bar to the left indicates 400 mu m.
Panel C, on the right, depicts Neodiplostomum leoni. It features a distinct separation between a pear-shaped anterior segment and a long, cylindrical posterior segment. The anterior segment contains the oral sucker, pharynx, ventral sucker, and a highly folded tribocytic organ. The posterior segment contains large, tandemly arranged testes and a central ovary. A scale bar to the left indicates 500 mu m.
Scanning electron photomicrographs of Neodiplostomum acatlotlii n. sp., (A) full body; (B) oral sucker; (C) ventral sucker; (D) holdfast organ; Neodiplostomum papaloapanensis n. sp., (E) full body; (F) oral sucker; (G) ventral sucker; (H) holdfast organ; Neodiplostomum leoni n. sp., (I) full body; (J) oral sucker; (K) ventral sucker; (L) holdfast organ.
Figure 3. Long description
A collection of twelve scanning electron photomicrographs arranged in three columns.
Left Column (Neodiplostomum acatlotlii n. sp.):
* Panel A: Full body view showing an elongated anterior and a bulbous posterior. Scale bar 500 micrometers.
* Panel B: Close-up of the oral sucker at the anterior tip. Scale bar 20 micrometers.
* Panel C: Circular ventral sucker with a central opening. Scale bar 20 micrometers.
* Panel D: Oval-shaped holdfast organ with radial striations. Scale bar 100 micrometers.
Middle Column (Neodiplostomum papaloapanensis n. sp.):
* Panel E: Full body view with a wider, heart-shaped anterior segment. Scale bar 400 micrometers.
* Panel F: Oral sucker showing a semi-circular opening. Scale bar 50 micrometers.
* Panel G: Ventral sucker with a horizontal slit-like opening. Scale bar 20 micrometers.
* Panel H: Holdfast organ with a distinct tri-lobed or clover-like appearance. Scale bar 100 micrometers.
Right Column (Neodiplostomum leoni n. sp.):
* Panel I: Full body view showing a segmented appearance with a distinct neck region. Scale bar 500 micrometers.
* Panel J: Oral sucker with a deep, V-shaped indentation. Scale bar 50 micrometers.
* Panel K: Ventral sucker with a thick, textured rim. Scale bar 50 micrometers.
* Panel L: Holdfast organ with deep central folds. Scale bar 50 micrometers.
Morphological comparison among the species of Neodiplostomum spp. Measurements in micrometres unless otherwise stated
Table 3. Long description
The table compares seven taxa: N. acatlotlii n. sp. (Mexico, n = 13), N. papaloapanensis n. sp. (Mexico, n = 9), N. leoni n. sp. (Mexico, n = 7), N. fastigatum (Brazil), N. lutzi (Brazil), N. nephrocystis (Brazil), and N. cornutum (Brazil).
Key morphological data (ranges with means in parentheses) include:
* Body Length (B L): Ranges from 0.762 mm in N. nephrocystis to 2.02 mm in N. leoni.
* Prosoma Length (P L): N. acatlotlii (692–1190) is significantly longer than N. nephrocystis (422–552).
* Prosoma Width (P W): N. papaloapanensis is the widest (525–761) compared to N. cornutum (219–296).
* Oral Sucker Length (O S L): N. leoni has the largest (103–132) while N. acatlotlii has the smallest (35–45).
* Ventral Sucker Width (V S W): N. leoni (100–174) is larger than N. acatlotlii (36–50).
* Holdfast Organ Length (H O L): N. papaloapanensis (189–327) is the largest, while N. nephrocystis (62–98) is the smallest.
* Testis Measurements: N. leoni shows high values for posterior testis length (P T L) at 273–342.
* Egg Dimensions: Egg length (E G L) is relatively consistent across species, ranging from 48 to 106, with N. lutzi and N. nephrocystis averaging 100.
Hosts include Rupornis magnirostris, Crotophaga sulcirostris, Pitangus sulfuratus, Crotophaga major, and Attila cinnamomeus.
BL, body length; PL, prosoma length; PW, prosoma width; OL, opistosoma length; OW, opistosoma width; PL/OL ratio, Prosoma:opisthosoma length ratio; OSL, oral sucker length; OSW, oral sucker width; VSL, ventral sucker length; VSW, ventral sucker width; OSW/VSW ratio, oral sucker width: ventral sucker width; PHL, pharynx length; PHW, pharynx width; HOL, Holdfast organ length; HOW, Holdfast organ width; ATL, anterior testis length; ATW, anterior testis width; PTL, posterior testis length; PTW, posterior testis width; OL, ovary length; OW, ovary width; EGL, egg length; EGW, egg width.
Remarks
Neodiplostomum acatlotlii n. sp., yielded morphological traits from the genus Neodiplostomum such as: Body distinctly bipartite, absence of pseudosuckers, presence of holdfast organ. Woodyard et al (Reference Woodyard, Rosser and Griffin2017) recognized eight species of Neodiplostomum associated with strigiform birds from the Americas, such as: N. americanum, N. buteonis, N. conicum, N. morenodathei, N. reflexum, N. microcotyle, N. accipitris, and N. travassosi. The new species differ in body shape and on several morphometric traits. For example, the new species differs in having a smaller body size (1.05–1.92 mm) than N. americanum (1.45–2.57 mm), N. buteonis (2.64–2.95 mm), and N. reflexum (1.10–3.59 mm). In addition, the new species also differs from N. travassosi and N. conicum in the opisthosoma length (349–806), vs (400–590) and (620–700) respectively, and prosoma/opisthosoma length ratio (1.47–1.98) vs (400–550) and (610–700) respectively. The new species also differs from N. accipitris in the prosoma size (692–1190 × 260–383) vs (840–1380 × 370–620), prosoma/opisthosoma length ratio (0.97–1.25) vs (0.39–0.50) in N. accipitris (see Dubois, Reference Dubois1970). The new species differs from N. morenodathei in the prosoma size (692–1190 × 260–383) vs (910–1540 × 540–1000), opisthosoma size (349–806 × 245–338) vs (680–1120 × 540–720) and oral sucker size (35–45 × 38–52) vs (48–72 × 48–72) (see Woodyard et al. Reference Woodyard, Rosser and Griffin2017). Finally, the new species differs from N. microcotyle in the prosoma size (692–1190 × 260–383) vs (390–550 × 200–300), prosoma/opisthosoma length ratio (1.47–1.98) vs (0.45–0.65).
Neodiplostomum papaloapanensis n. sp.
Type-host: Crotophaga sulcirostris Swainson, 1827.
Site of infection: Intestine
Type locality: Tlacotalpan, Veracruz, Mexico (18° 37′ 24.8″ N 95° 39′ 17.24″ W).
Type material: CNHE: 12592 (holotype); 12593 (paratypes).
Representative DNA sequences: PZ535836-837 (SSU); PZ535821-822 (LSU); PZ535829-830 (ITS); PZ535949-950 (cox 1).
Etymology: The specific epithet refers to the Papaloapan River, which means river of butterflies in the Náhuatl language, where the type host inhabits.
Description: Description (based on nine adult specimens plus two specimens for SEM); measurements of holotype (Figures 2B and 3E–H) given in text; measurements of the entire series given in Table 3. Body distinctly bipartite oval, with a rounded posterior end, 1.13 mm long. Prosoma oval, 567 long, covered with tiny spines, widest at the level of the holdfast organ 562. Opisthosoma oval, unarmed, 563 long, narrower than prosoma, 562 wide. Prosoma-opisthosoma length ratio 1.08. Oral sucker, terminal muscular, subspherical 49 × 33, surrounding with small dome-like papillae. Ventral sucker oval, muscular, papillae absent, larger than oral sucker 83 × 116. Oral sucker: ventral sucker width ratio 0.28. Holdfast organ, laterally protruding, immediately posterior to ventral sucker, oval, muscular, 220 × 185. Proteolytic gland dorsal to the posterior part of the holdfast organ. Prepharynx absent. Pharynx muscular, oval, 52 × 42. Oesophagus absent. Caeca bifurcation in the most anterior quarter of the prosoma. Caeca slender, reaching the posterior end of the body. Testes two, tandem, contiguous, slightly separated. Anterior testis asymmetrical 260 × 190. Posterior testis bilobed, transversely elongated, 272 × 330. Seminal vesicle not observed. Ovary ovoid, placed between the anterior testis and the prosoma-opisthosoma junction 99 × 59. Vitellarium located from near the caecal bifurcation in the prosoma, extending to the opisthosoma to the posterior margin of the testis. Egg, 99 × 59. Excretory pore terminal.
Remarks: Neodiplostomum papaloapanensis n. sp., exhibited morphological traits from the genus Neodiplostomum such as: Body distinctly bipartite, absence of pseudosuckers, presence of holdfast organ. The new species has a unique combination of morphological traits that have not been observed in other species of Neodiplostomum. To date, two species (N. nephrocystis and N. ellipticum) have been recorded in cuculiform birds from the Americas. The new species differ in body shape, prosoma and opisthosoma oval vs prosoma elongate-oval and opisthosoma subcylindrical on N. nephrocystis and prosoma oval and opisthosoma cylindrical on N. ellipticum. Additionally, the new species differs morphometrically from N. ellipticum in having a smaller body size (1.13–1.31 mm) vs (1.55 mm) on N. ellipticum; opisthosoma size (563–717 × 404–632) on Neodiplostomum papaloapanensis n. sp., vs (300–800 × 300–500) on N. ellipticum. The new species has a smaller oral sucker (44–59 × 33–73), vs (37–86 × 38–97) on N. ellipticum; a larger holdfast organ (189–327) on Neodiplostomum papaloapanensis n. sp., vs (135–300) N. ellipticum; anterior testis size (197–465 × 190–489) on Neodiplostomum papaloapanensis n. sp., vs (170–280 × 220–435) N. ellipticum; posterior testis (176–516 × 280–587) on Neodiplostomum papaloapanensis n. sp., vs (180–260 × 340–450) on N. ellipticum; ovary size (123–207 × 53–97) on Neodiplostomum papaloapanensis n. sp., vs (54–95 × 140–180) on N. ellipticum.
Neodiplostomum leoni n. sp.
Type-host: Pitangus sulphuratus Linnaeus, 1766.
Site of infection: Intestine
Type locality: Tlacotalpan, Veracruz, Mexico (18° 37′ 24.8″ N 95° 39′ 17.24″ W).
Type material: CNHE: 12594 (holotype); 12595 (paratypes).
Representative DNA sequences: PZ535834-835 (SSU); PZ535823-824 (LSU); PZ535831-832 (ITS); PZ535951-952 (cox 1).
Etymology: The species is named in honour of Dr. Gerardo Pérez Ponce de León (Universidad Nacional Autónoma de México) for his remarkable contributions to the taxonomy, ecology, and systematics of digeneans in the Neotropic.
Description (Figures 2C and 3I–L)
Description (based on seven adult specimens plus two specimens for SEM); measurements of holotype given in text; measurements of the entire series given in Table 3. Body distinctly bipartite, with a rounded posterior end, 1.83 mm long. Prosoma elongated-oval, 882 long, covered with tiny spines, ventrally concave posterior to ventral sucker; widest at the level of the holdfast organ 356. Opisthosoma cylindrical, unarmed, 951 × 322. Prosoma-opisthosoma length ratio 1.07. Oral sucker terminal, subspherical, 199 × 120, strongly muscular, funnel-shaped, with two well-defined subconical lobes, one on each side. Ventral sucker muscular, smaller than the oral sucker, 112 × 115, surrounding glandular part of small gland-cells and tiny spines. Oral sucker: ventral sucker width ratio 1.04. Holdfast organ immediately posterior to ventral sucker, oval transversely elongated with ventral muscular portion 174 × 108. Proteolytic gland dorsal to the posterior part of the holdfast organ. Prepharynx absent. Pharynx muscular oval 92 × 41. Oesophagus absent. Caeca bifurcation in the most anterior quarter of the prosoma. Caeca slender, reaching the posterior end of the body. Testes two, tandem, contiguous, separated. Anterior testis asymmetrical 269 × 163. Posterior testis somewhat bilobed, 282 × 232. Seminal vesicle not observed. Ovary ovoid, placed between the anterior testis and the prosoma-opisthosoma junction, 99 × 79. Vitellarium located from near the caecal bifurcation in the prosoma, extending to the opisthosoma to the posterior margin of the body. Egg, 1, 96 × 51. Excretory pore terminal.
Remarks: Neodiplostomum leoni n. sp. contains morphological characteristics belonging to the genus Neodiplostomum such as: Body distinctly bipartite, absence of pseudosuckers, presence of holdfast organ, prosoma concave, and vitellarium on both sides of the body. The new species can be related to the other three congeneric species, such as N. fastigatum, N. cornutum, and N. nephrocystis, in having an apical structure and could be a synapomorphic trait. However, Neodiplostomum leoni n. sp. differs from N. fastigatum and N. cornutum in having two muscular lobes, one on each side of the oral sucker, vs apical structure in the form of fleshy extending about mid-length of the oral sucker, forming a pre-oral lobe on N. fastigatum, and three well-developed muscular lobes and a fleshy tegumental hood around the oral sucker on N. cornutum. In addition, Neodiplostomum leoni n. sp., resembles N. nephrocystis in the apical structure. However, the new species has two well-defined subconical lobes vs apical structure with flanges on N. nephrocystis. Besides the differences in the shape of the apical structure, the new species can also be distinguished morphometrically from N. nephrocystis in all traits analysed in this study, because the specimens of Neodiplostomum leoni n. sp. are larger than N. nephrocystis, collected by Achatz et al (Reference Achatz, Kostadinova, Georgieva, Fecchio and Tkach2025) from Brazil (see Table 3). The morphometrical traits of Neodiplostomum leoni n. sp., also were larger than N. nephrocystis reported by Dubois (1938, 1970); for example, body size (1.68–2.02) vs (1.5–1.8); oral sucker width (107–130) vs (100–120); ventral sucker (89–127 × 100–174) vs 77–91 × 93–112); pharynx (66–92 × 41–50) vs (60–80 × 38–48); anterior testis (236–322 × 129–270) vs (160–215 × 190–250); posterior testis (273–342 × 171–357) vs (170–235 × 215–285) and ovary length (77–109) vs (72–95).
Phylogenetic analyses
Nuclear markers
The five newly generated (SSU) sequences were analysed together with 11 sequences of Neodiplostomum spp. plus 15 sequences of other diplostomids used as outgroups (Table 2). The alignment comprised 31 sequences with 1,717 characters. The phylogenetic analyses identified two independent major clades unrelated to Neodiplostomum with strong bootstrap support (100%) and a strong Bayesian posterior probability (1.0) (Figure 4). The first major clade contained sequences of two species, N. banghami (OL799087) and N. americanum (KY851307). The second major clade included sequences of 10 species of Neodiplostomum spp., with strong Bayesian posterior probabilities (1.0) and within it, four well-supported subclades. The first contained the species N. spathula (Creplin, 1829) La Rue, 1926 (MK089351), plus two isolates of N. cratera (OL799071, OL799074), N. lucidum (OL799076), and N. reflexum (OL799089), in an unresolved branch (Figure 4). The second subclade contained N. attennuatum (von Linstow, 1906) La Rue, 1926 (MG770033) at the base of the node, plus Neodiplostomum sp. (OL799096), N. vaucheri (OL799095), N. microcotyle (OL799079), and the new species described herein as Neodiplostomum acatlotlii n. sp. from the roadside hawk, R. magnirostris. The third subclade was formed by two isolates of the second new species described herein as Neodiplostomum papaloapanensis n. sp, from the groove-billed ani, C. sulcirostris. Finally, the fourth subclade was formed by two isolates of the third new species, Neodiplostomum leoni n. sp., from the great kiskadee, P. sulphuratus, with strong bootstrap support (100%) and a strong Bayesian posterior probability (1.0) (Figure 4).
Phylogenetic trees inferred with maximum likelihood (ML) and consensus Bayesian inference (BI) of SSU from nuclear ribosomal DNA. Numbers near internal nodes show maximum likelihood bootstrap percentage values and Bayesian posterior probabilities. Sequences generated in this study are in bold.
Figure 4. Long description
A rectangular cladogram rooted on the left with Cyathocotyle prissica M H 5 2 1 2 4 9. The tree branches into several major clades.
Top Clade: Includes Hysteromorpha triloba and Alaria species.
Middle Clades: Feature Diplostomum, Austrodiplostomum, Tylodelphys, and Neodiplostomum species.
Bottom Clade: Contains the sequences generated in this study, highlighted in bold and shaded in gray boxes. These include Neodiplostomum leoni n. sp. P Z 5 3 5 8 3 4 and P Z 5 3 5 8 3 5, Neodiplostomum papaloapanensis n. sp. P Z 5 3 5 8 3 6 and P Z 5 3 5 8 3 7, and Neodiplostomum acatlotlii n. sp. P Z 5 3 5 8 3 3.
Internal nodes are labeled with bootstrap values and posterior probabilities, such as 100/1 or 99/1. To the right of the terminal taxa, black silhouettes represent host animals, including various birds and mammals. A legend on the bottom left identifies these silhouettes as Falconidae, Accipitridae, Strigidae, Cuculidae, Tyrannidae, Phyllostomidae, Didelphidae, and Mustelidae. A scale bar at the bottom indicates 0.02 substitutions per site.
The eight newly generated (LSU) sequences were analysed together with 16 sequences of Neodiplostomum spp. plus 15 sequences of other diplostomids used as outgroups (Table 2). The alignment comprised 39 sequences with 1,134 characters. The phylogenetic analyses also identified two independent major clades from Neodiplostomum with strong bootstrap support (100%) and a strong Bayesian posterior probability (1.0) (Figure 5). The first major clade also contained sequences of two species, N. banghami (OL799087) and N. americanum (KY851307), plus Sphincterodiplostomum musculosum Dubois, 1938 from Diplostomidae (Figure 7). The second major subclade contained the remain sequences of Neodiplostomum spp., which were subdivided into four subclades. The first subclade contained a single sequence of Neodiplostomum sp. (OL799096), one sequence of N. vaucheri (OL799108), another of N. microcotyle (OL799079), plus four sequences of the new species Neodiplostomum acatlotlii n. sp., from the roadside hawk, R. magnirostris. The second subclade contained two sequences of N. cratera (OL799097, OL799099), one sequence of N. reflexum (OL799089), one of N. lucidum (OL799102), and at the base of the branch, the sequence of N. seoulense (Seo, Rim and Lee, 1964) (OP185288). The third subclade contained two sequences of N. cornutum (PP848960, PP848961), one sequence of N. nephrocystis (PP848965), one of N. fastigatum (PP848963), plus two sequences of the new species Neodiplostomum leoni n. sp. from the great kiskadee, P. sulphuratus, with strong bootstrap support (100%) and a strong Bayesian posterior probability (1.0) (Figure 5). Finally, the fourth subclade was formed for one sequence of N. ellipticum (PP848962), plus two sequences of the new species described herein as Neodiplostomum papaloapanensis n. sp., from the groove-billed ani, C. sulcirostris (Figure 5).
Phylogenetic trees inferred with maximum likelihood (ML) and consensus Bayesian inference (BI) of LSU from nuclear ribosomal DNA. Numbers near internal nodes show maximum likelihood bootstrap percentage values and Bayesian posterior probabilities. Sequences generated in this study are in bold.
Figure 5. Long description
A rectangular cladogram rooted by M K 6 5 0 4 5 0 Suchocyathocotyle crocodilii at the top. The tree branches into several major clades.
Top clades include Alaria americana, Austrodiplostomum compactum, and Diplostomum species.
A middle clade contains Posthodiplostomum and Uvulifer species.
The largest lower clade focuses on Neodiplostomum. Within this group, three new species are highlighted in bold and shaded gray boxes:
1. Neodiplostomum papaloapanensis n. sp. (P Z 5 3 5 8 2 1 and P Z 5 3 5 8 2 2) clustered with a 9 9 forward slash 1 support value.
2. Neodiplostomum leoni n. sp. (P Z 5 3 5 8 2 3 and P Z 5 3 5 8 2 4) clustered with an 8 9 forward slash 1 support value.
3. Neodiplostomum acatlotlii n. sp. (P Z 5 3 5 8 1 7 through P Z 5 3 5 8 2 0) at the bottom of the tree with a 9 8 forward slash 1 support value.
To the right of the taxa names, black silhouettes represent host animals including snakes, owls, various birds, bats, procyonids, and rodents. A legend on the bottom-left identifies these host families: Accipitridae, Strigidae, Cuculidae, Tyrannidae, Phyllostomidae, Procyonidae, Mustelidae, Muridae, and Colubridae. A scale bar at the bottom indicates 0.05 substitutions per site.
The eight newly generated (ITS) sequences were analysed together with 16 sequences of Neodiplostomum spp. plus nine sequences of other diplostomids used as outgroups (Table 2). The alignment comprised 33 sequences with 1,122 characters. The phylogenetic analyses showed Neodiplostomum as monophyletic with moderate bootstrap support and Bayesian posterior probability (Figure 6). This tree showed several major subclades. The first subclade contained sequences of two species, N. banghami (OL799087) plus N. americanum (JF769485), which are sister to five sequences of N. americanum (OL799085, OL799086, KY851307-309). The second subclade was formed for two sequences of the new species Neodiplostomum leoni n. sp. from the great kiskadee, P. sulphuratus, plus another two sequences of Neodiplostomum papaloapanensis n. sp., from the groove-billed ani, C. sulcirostris, with strong bootstrap support (100%) and a strong Bayesian posterior probability (1.0) (Figure 6). The third and fourth subclades were formed by a single sequence of Neodiplostomum sp. (OL799096) and N. vaucheri (OL799095), respectively. The fifth subclade was formed for four sequences of the new species Neodiplostomum acatlotlii n. sp., from the roadside hawk, R. magnirostris. The sixth subclade contained a single sequence of N. microcotyle (OL799079). Finally, the seventh subclade was formed for two sequences of N. reflexum (OL799093, OL799094), two sequences of N. lucidum (OL799075, OL799078), and two sequences of N. cratera (OL799071, OL799074) with moderate bootstrap support (100%) and Bayesian posterior probability (see Figure 6).
Phylogenetic trees inferred with maximum likelihood (ML) and consensus Bayesian inference (BI) of ITS1–5.8S–ITS2 from nuclear ribosomal DNA. Numbers near internal nodes show maximum likelihood bootstrap percentage values and Bayesian posterior probabilities. Sequences generated in this study are in bold.
Figure 6. Long description
A phylogenetic tree diagram with horizontal branches.
* Root and Outgroups: The tree originates from a root on the left. The top-most branches include Alaria americana and Uvulifer spinatus.
* Major Clades:
- A clade containing Diplostomum mergi and Diplostomum species with bootstrap/posterior probability values of 100/1 and 99/0.94.
- A clade containing Austrodiplostomum compactum and Tylodelphys species.
- A large clade of Neodiplostomum species. Within this, three groups are highlighted in gray boxes: Neodiplostomum papaloapanensis n. sp. (P Z 5 3 5 8 2 9, P Z 5 3 5 8 3 0), Neodiplostomum leoni n. sp. (P Z 5 3 5 8 3 1, P Z 5 3 5 8 3 2), and Neodiplostomum acatlotlii n. sp. (P Z 5 3 5 8 2 5 through P Z 5 3 5 8 2 8).
* Node Labels: Numerical values like 100/1, 96/1, and 76/0.99 are placed at internal nodes.
* Host Silhouettes: To the right of the terminal taxa, a vertical column of animal silhouettes (birds, bats, and small mammals) indicates the host for each sequence.
* Legend: In the bottom left, a key matches silhouettes to family names: Laridae, Falconidae, Accipitridae, Strigidae, Cuculidae, Tyrannidae, Ranidae, Phyllostomidae, Didelphidae, and Mustelidae.
* Scale Bar: A horizontal line at the bottom left indicates a genetic distance of 0.06.
Mitochondrial marker
The ten newly generated (cox 1) sequences were analysed together with 36 sequences of Neodiplostomum spp. plus two sequences of other diplostomids used as outgroups (Table 2). The alignment comprised 48 sequences with 403 characters and included the barcoding region from the cox 1. The phylogenetic analyses showed Neodiplostomum paraphyletic, with low bootstrap support and a low Bayesian posterior probability (Figure 7). This tree showed that two sequences of N. banghami (OL770054, OL770055) are sister taxa of four sequences of N. americanum (OL770046-048, OL770051). This tree also revealed that three sequences of Neodiplostomum papaloapanensis n. sp. from the groove-billed ani, C. sulcirostris, and common squirrel-cuckoo, P. cayana, are sister to two sequences of N. ellipticum (PP844930, PP844930). Another clade was formed with two sequences of N. lutzi (PP844934, PP844935), which are sister to a clade formed for three sequences of Neodiplostomum leoni n. sp. from the great kiskadee, P. sulphuratus, and boat-billed flycatcher, M. pitangua, plus a single sequence of N. nephrocystis (PP844936), two sequences of N. cornutum (PP844928, PP844929), and two sequences of N. fastigatum (PP844932, PP844933) with moderate posterior probability and low bootstrap support values. Another clade was formed by two sequences of N. microcotyle (OL770044, OL770045), plus four sequences of the new species Neodiplostomum acatlotlii n. sp., from the roadside hawk, R. magnirostris. This clade is sister to remains species of Neodiplostomum analysed in this study. For example, Neodiplostomum sp. (OL770068) was placed unresolved node of the three, whereas the two sequences of N. vaucheri (OL770065, OL770067) formed a subclade, which is sister to the unresolved clade formed for nine sequences of N. cratera (OL770020-021, OL770023-027, OL770029-030) and four sequences of N. lucidum (OL770031, OL770037-039), plus three sequences of N. reflexum (OL770057-058, OL770060) (Figure 7).
Phylogenetic trees inferred with maximum likelihood (ML) and consensus Bayesian inference (BI) mitochondrial cytochrome c oxidase subunit 1 (cox 1) genes. Numbers near internal nodes show maximum likelihood bootstrap percentage values and Bayesian posterior probabilities. Sequences generated in this study are in bold.
Figure 7. Long description
A phylogenetic tree rooted with M K 1 7 2 7 9 2 Tylodelphys azteca at the top left. The tree branches into several major clades of Neodiplostomum species.
* The top clade includes Neodiplostomum banghami, N. americanum, Hysteromorpha triloba, and N. ellipticum.
* A highlighted gray box contains the bolded sequences for Neodiplostomum papaloapanensis n. sp. (P Z 5 3 7 0 4 2, P Z 5 3 5 9 4 9, P Z 5 3 5 9 5 0).
* Below this, another gray box highlights Neodiplostomum leoni n. sp. (P Z 5 3 7 0 4 3, P Z 5 3 5 9 5 1, P Z 5 3 5 9 5 2).
* Further down, a third gray box highlights Neodiplostomum acatlotlii n. sp. (P Z 5 3 5 9 4 8, P Z 5 3 5 9 4 6, P Z 5 3 5 9 4 5, P Z 5 3 5 9 4 7).
* The bottom half of the tree includes N. vaucheri, N. cf. cratera, N. cf. lucidum, and N. reflexum.
To the right of each terminal taxon is a silhouette representing the host animal, including various birds, snakes, and mammals. A legend at the bottom left maps these silhouettes to family names: Colubridae, Falconidae, Accipitritidae, Strigidae, Cuculidae, Tyrannidae, Ranidae, Phyllostomidae, Procyonidae, Didelphidae, and Mustelidae. Support values (M L bootstrap/B I posterior probability) are provided at nodes, such as 100/1 or 98/0.99. A scale bar representing 0.05 substitutions per site is at the bottom.
In summary, the phylogenetic analyses inferred with four data sets (three nuclear and one mitochondrial molecular markers) are congruent in that the three new species, described herein, such as Neodiplostomum acatlotlii n. sp., Neodiplostomum papaloapanensis n. sp., and Neodiplostomum leoni, associated with birds from the Neotropical region of Mexico, formed three independent lineages, supporting their morphological differences. In addition, the genetic divergence estimated among the three new species was high with all the molecular markers analyses; for example, the interspecific genetic divergence ranged from 1.0% to 1.29% with SSU, from 2.92% to 3.54% with LSU, from 6.05% to 9.45% with ITS, and finally, with cox 1 was from 13.68 to 17.48%. The largest interspecific genetic divergence found with each molecular marker of the three new species with described species (excepting N. banghami and N. americanum, which were nested in an independent clade from remains species of Neodiplostomum) was between Neodiplostomum leoni n. sp. and N. vaucheri, with 1.47% on SSU, between Neodiplostomum acatlotlii n. sp., N. fastigatum, N. nephrocystis was 3.63% with LSU, between Neodiplostomum papaloapanensis n. sp. and N. vaucheri was 9.44% with ITS, between Neodiplostomum papaloapanensis n. sp. and N. cratera was 18.7% with cox 1. Finally, the lowest genetic divergence found among the four molecular markers was between Neodiplostomum acatlotlii n. sp., and N. vaucheri with 0.24 % for SSU, between Neodiplostomum papaloapanensis n. sp. and N. ellipticum was 0.27 % with LSU, between Neodiplostomum acatlotlii n. sp. and N. microcotyle was 2.89% with ITS, and between Neodiplostomum leoni n. sp. and N. nephrocystis was 3.68% with cox 1 (Supplementary Tables S4–S7).
Discussion
The genus Neodiplostomum was erected to include Neodiplostomum spathulaeforme (Brandes, 1888), a parasite of the long-eared owl, Asio otus L., from Europe, and since then, the taxonomic history, species composition, and systematics of Neodiplostomum have been complex and unsettled because of the phenotypic plasticity of its morphological traits (Achatz et al 2022, 2025; Cacique et al Reference Cacique, Barriga, Bello, Pino, Salcedo-Mejia, Ñacari, Crucces, Mondragón-Martínez and Chero2025). The genetic library of Neodiplostomum spp. has recently increased and provides a great opportunity to clarify the taxonomy and species composition. Recent taxonomic studies that combined both morphological and molecular data have started to clarify the taxonomy of the species, and new species have been described (Achatz et al 2022, Reference Achatz, Kostadinova, Georgieva, Fecchio and Tkach2025; Cacique et al Reference Cacique, Barriga, Bello, Pino, Salcedo-Mejia, Ñacari, Crucces, Mondragón-Martínez and Chero2025; Woodyard et al Reference Woodyard, Rosser and Griffin2017). Recent molecular evidence suggests that Neodiplostomum is paraphyletic (see Achatz et al 2022, Reference Achatz, Kostadinova, Georgieva, Fecchio and Tkach2025; Heneberg et al Reference Heneberg, Sitko and Těšínský2020). However, these studies lacked genetic information on the type species, which is mandatory for better understanding and clarifying the systematics of Neodiplostomum. Our phylogenetic analyses indicated that the small and large subunits from nuclear DNA agreed with previously published phylogenies because the species N. banghami (OL799087, SSU; OL799105, LSU) and N. americanum (KY851307, SSU; OL799105, LSU) were placed in an independent clade from their congeneric species (see Achatz et al 2022, 2025; Heneberg et al Reference Heneberg, Sitko and Těšínský2020). In the current study, we combined morphological characteristics and generated genetic information from four molecular markers to support the morphological differences found among the three new species. The phylogenetic analyses inferred that each molecular marker supported the monophyly of three subclades, representing the three new species described herein. However, the topologies and systematic positions of the three new species differed across the analyses because different taxa were used (see Figures 4–7). In particular, cox 1 was the molecular marker with the highest rate of substitution compared with the nuclear molecular markers, such as SSU, LSU, and ITS. It is well known that cox 1 is an excellent molecular marker for delineating species, as it has been used in the detection of cryptic species and population genetics in diplostomids (Achatz et al 2022, Reference Achatz, Kostadinova, Georgieva, Fecchio and Tkach2025; Locke et al Reference Locke, McLaughlin and Marcogliese2010, Reference Locke, Al-Nasiri, Caffara, Drago, Kalbe, Lapierre, McLaughlin, Nie, Overstreet, Souza, Takemoto and Marcogliese2015; López-Jiménez et al Reference López-Jimenez, Pérez-Ponce de León and García-Varela2017, Reference López-Jimenez, Hernández-Mena, Solórzano-García and García-Varela2022, Reference López-Jiménez, González-García, Andrade-Gómez and García-Varela2023; Sereno-Uribe et al Reference Sereno-Uribe, Andrade-Gómez, Ostrowski de Núñez, de León G and García-Varela2019; Woodyard et al Reference Woodyard, Rosser and Griffin2017), whereas SSU and LSU are excellent nuclear molecular markers for delineating the genera from Diplostomidae (Achatz et al 2022; Hernández-Mena et al Reference Hernández-Mena, García-Prieto and García-Varela2014, Reference Hernández-Mena, García-Varela and Pérez-Ponce de León2017; Tkach et al Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajan and Melo2020). In addition, the genetic divergence estimated with cox 1 among the three new species described herein (Neodiplostomum spp.) ranged from 13.68% to 17.48%; these levels of genetic divergence are greater than those previously recognised by Achatz et al (2022, Reference Achatz, Kostadinova, Georgieva, Fecchio and Tkach2025), which ranged from 8.6% to 13.4% and from 7.6% to 15.6%, respectively. Interestingly, we detected low values of genetic divergence among Neodiplostomum leoni n. sp. from the great kiskadee, P. sulphuratus, and the boat-billed flycatcher, M. pitangua and N. nephrocystis from the great kiskadee, P. sulphuratus from Mato Grosso, Brazil, ranging from 3.68% to 3.75%. Cacique et al (Reference Cacique, Barriga, Bello, Pino, Salcedo-Mejia, Ñacari, Crucces, Mondragón-Martínez and Chero2025) compared the morphological and molecular characteristics of specimens of N. vaucheri recovered from a fringe-lipped bat, Trachops cirrhosis Gray, from two countries, Ecuador and Peru. Their study revealed morphometric differences that were corroborated by genetic differences of up to 4%, suggesting that the specimens from Ecuador could represent a cryptic species. In addition, Achatz et al (2022) reported morphological differences between N. cratera from the northern USA and N. laurei (synonymised with N. cratera) from the southern USA, which should be reassessed because both samples exhibited genetic divergence ranging from 2.6% to 2.9% with cox 1.
The results of the present study revealed that the three new species in the genus Neodiplostomum, increase its diversity in the Neotropics and provide a phylogenetic framework for exploring the ecological associations among the parasites and their definitive hosts. For example, the phylogenetic analyses inferred from each molecular marker supported the close relationship of Neodiplostomum acatlotlii n. sp. from the roadside hawk, R. magnirostris, with N. microcotyle, which was described from the great black hawk, Buteogallus urubitinga Gmelin, from Brazil (Dubois, Reference Dubois1970) and recently recorded in the black-collared hawk, Busarellus nigricollis Latham from the Pantanal, Brazil (Achatz et al 2022), suggesting an ecological association of both species of neodiplostomids with accipitrid birds. In addition, the results of the phylogenetic analyses inferred from the LSU and cox 1 datasets revealed that Neodiplostomum leoni n. sp. from the great kiskadee, P. sulphuratus, and the boat-billed flycatcher, M. pitangua, which is closely related to N. nephrocystis, N. cornutum, and N. fastigatum, parasitised tyranid birds from the Neotropics, supporting an ecological association with this group of birds. Finally, the third species, named Neodiplostomum papaloapanensis n. sp., recovered from two cuculid bird species, i.e., the groove-billed ani, C. sulcirostris, and the common squirrel-cuckoo, P. cayana, yielded a close relationship in the LSU and cox 1 trees with N. ellipticum and N. lutzi associated with the smooth-billed ani, Crotophaga ani L, supporting an ecological relationship with cuckoos from South America. The ecological associations detected in this study are in agreement with those reported by Achatz et al (Reference Achatz, Kostadinova, Georgieva, Fecchio and Tkach2025), who reported that species of Neodiplostomum experienced multiple host-switching events between birds of prey and mammals and between cuckoos and passerines.
The current evidence suggests that neodiplostomid species use different ways to complete their life cycles; i.e., Neodiplostomum acatlotlii n. sp. was found in the roadside hawk, R. magnirostris. This carnivorous bird feeds on a wide range of prey, such as small vertebrates (lizards, frogs, and rodents) and invertebrates (mainly insects) (Gomes and Tubelis Reference Gomes and Tubelis2022; Panasci and Whitacre Reference Panasci and Whitacre2000), which could serve as second intermediate hosts (Blasco-Costa and Locke Reference Blasco-Costa, Locke, Rollinson and Stothard2017; Izrailskaia and Besprozvannykh Reference Izrailskaia and Besprozvannykh2023). The species Neodiplostomum papaloapanensis n. sp. was found in two cuckoo bird species, the groove-billed ani, C. sulcirostris, and the common squirrel-cuckoo, P. cayana, which feed on small vertebrates (lizards) and invertebrates (insects, grasshoppers, dragonflies, caterpillars, and beetles) (Alcántara et al Reference Alcántara, Velásquez and Guzmán2012; Julián-Caballero Reference Julián Caballero2023; van Dort et al Reference Van Dort, Kirwan, Fitzgerald, Schulenberg and Billerman2025); the invertebrates that have an aquatic phase could serve as second intermediate hosts. Finally, the species Neodiplostomum leoni n. sp. was found in two flycatcher species, the great kiskadee, P. sulphuratus, and the boat-billed flycatcher, M. pitangua. Both bird species feed on invertebrates, such as insects, dragonflies, caterpillars, beetles, and freshwater snails, and occasionally small vertebrates, such as lizards and frogs, which can serve as second intermediate hosts (Brush and Fitzpatrick Reference Brush, Fitzpatrick, Poole and Gill2020; Mobley Reference Mobley, del Hoyo, Elliott, Sargatal, Christie and de Juana2020).
The three new species described herein were found in three bird families, Accipitridae, Tyrannidae, and Cuculidae, which are distributed sympatrically in the Veracruzan biogeographical province in the Neotropical region of Mexico. This province holds 701 bird species, including 357 species belonging to Passeriformes, 8 to Cuculiformes and 33 to Accipitriformes; considering that few bird species have been explored from a helminthological perspective, the likelihood of describing new species is high because the biodiversity of potential definitive hosts is even greater, given that 23 species of Passeriformes are endemic to this biogeographical province (Berlanga et al Reference Berlanga, Gómez de Silva, Vargas-Canales, Rodriguez-Contreras, Sánchez-Gonzalez, Ortega-Alvarez and Calderon-Parra2015; Navarro-Singüenza et al Reference Navarro-Sigüenza, Rebón-Gallardo, Gordillo-Martínez, Peterson, Berlanga-García and Sánchez-González2013).
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X26101849.
Acknowledgements
We thank Laura Márquez and Nelly López Ortiz from LaNabio for their help during the sequencing of the DNA fragments. We also thank Berenit Mendoza for her help with the use of the SEM unit. MTGG and YAP thank the support of the Programa de Posgrado en Ciencias Biologicas, UNAM, and Secretaría de Ciencia, Humanidades, Tecnología e Innovación (SECIHTI) for funding and for the support of this research through their scholarship to MTGG (CVU 956064) and YAP (CVU 1254979).
Author’s contribution
ALSU, MTGG, YAP, MPOO, and MGV conceived and designed the study. ALSU, MTGG, YAP, and MPOO conducted data gathering. ALSU and MTGG performed phylogenetic analyses. ALSU, MTGG, YAP, MPOO, and MGV wrote and edited the article.
Competing interests
No conflict of interest exists among the authors.
Ethical approval
The sampling in this work complies with the current laws and animal ethics regulations of Mexico. Specimens were collected under the Cartilla Nacional de Colector Científico (FAUT 0202) issued by the Secretaría del Medio Ambiente y Recursos Naturales (SEMARNAT) to M.G.V.
Consent to participate
All the listed authors have made significant contributions to the study and agreed to participate.
Consent for publication
All authors have read and agreed to the published version of the manuscript.
Financial support
This research was supported by the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT-UNAM) IN204425 and Secretaría de Ciencia, Humanidades, Tecnología e Innovación (SECIHTI), (CBF-2025) No. 1433 to MGV.
MTGG and YAP thank the Posgrado en Ciencias Biologicas, UNAM, and Consejo Nacional de Humanidades, Ciencias y Tecnologías CONAHCYT for funding and for the support of this research through their scholarship to MTGG and YAP.
Open access
