Introduction
Dicrocoeliidae Looss, 1899 is a family of digenean parasites from the bile ducts, gallbladder and intestines of birds and, rarely, mammals distributed around the world, including approximately 400 species classified into 46 genera (Hildebrand et al., Reference Hildebrand, Sitko, Zaleśny, Jeżewski and Laskowski2016, Reference Hildebrand, Pyrka, Sitko, Jeżewski, Zaleśny, Tkach and Laskowski2019; Tkach et al., Reference Tkach, Achatz, Hildebrand and Greiman2018). The genus Lyperosomum Looss, 1899 is among the most diverse genera in this family, with approximately 33 recognized species, mostly parasitizing passerine birds (Hildebrand et al., Reference Hildebrand, Pyrka, Sitko, Jeżewski, Zaleśny, Tkach and Laskowski2019). The species of Lyperosomum are characterized by the following traits: oral sucker smaller than the ventral sucker, testes positioned closely to the ventral sucker, ovary posterior and distant from the posterior testis, genital pore located anterior to the intestinal bifurcation and vitellarium forming two relatively long lateral bands of follicles, beginning at the level of the testes and not reaching the caecal ends (Pojmańska, Reference Pojmańska, Bray, Gibson and Jones2008). Based on these morphological traits, the history of the taxonomy and species composition of the genus Lyperosomum has been complex and unstable due to the phenotypic plasticity of some diagnostic characteristics that define the species. Recently, Hildebrand et al. (Reference Hildebrand, Pyrka, Sitko, Jeżewski, Zaleśny, Tkach and Laskowski2019) conducted one of the most extensive studies of the genus Lyperosomum, combining morphological and molecular data. Their analyses also included species representing the genera Skrjabinus Bhalerao, 1936 and Zonorchis Travassos, 1944 from Dicrocoeliidae. These authors found that the species of Lyperosomum analysed were paraphyletic because some species of Zonorchis were nested in the genus Lyperosomum.
In the Americas, four species of the genus Lyperosomum have been recorded. Lyperosomum intermedium Denton & Kinsella, Reference Denton and Kinsella1972 was described from the pancreas of rice rats, Oryzomys palustris Harlan, 1837, from Georgia and Florida in the US (Denton & Kinsella, Reference Denton and Kinsella1972). Lyperosomum petiolatum (Railliet, 1900) Hildebrand, Pyrka, Sitko, Jeżewski, Zaleśny, Tkach & Laskowski, Reference Hildebrand, Pyrka, Sitko, Jeżewski, Zaleśny, Tkach and Laskowski2019 was isolated from the gall bladder of blue jays, Cyanocitta cristata (Linnaeus, 1758), from Texas, Mississippi and Nebraska in the USA (Denton & Byrd, Reference Denton and Byrd1951). Lyperosomum oswaldoi Travassos, 1919 was described from the liver and gall bladder of brown thrashers, Toxostoma rufum (Linnaeus, 1758), from Georgia, Mississippi and Texas in the USA (Denton & Byrd, Reference Denton and Byrd1951) and in the bile duct of great antshrike, Taraba major (Vieillot, 1816) from Argentina (Travassos, Reference Travassos1944; Lunaschi & Drago, Reference Lunaschi and Drago2013). Finally, Lyperosomum byrdi Denton & Krissinger, Reference Denton and Krissinger1975 was described from the liver and gall bladder of rufous-sided towhees, Pipilo erythrophthalmus (Linnaeus, 1758), from Florida and Georgia in the USA (Denton & Krissinger, Reference Denton and Krissinger1975).
During a helminthological expedition in northern Mexico, adult digeneans were recovered from the intestine of the golden-fronted woodpecker, Melanerpes aurifrons (Wagler, 1829). The examination of this material revealed the presence of an undescribed species of the genus Lyperosomum. Therefore, the aim of this study was to (1) provide a morphological description of the new species and (2) test the systematic position of the new species within Lyperosomum using molecular data from the large subunit (LSU) of nuclear ribosomal DNA and cytochrome c oxidase subunit 1 (cox 1) from mitochondrial DNA.
Materials and methods
Specimen collection
A total of 12 specimens of Lyperosomum sp. were obtained from a single M. aurifrons individual from northern Mexico. The host was examined for parasites under a dissecting microscope a few hours after its capture. The collected digeneans were preserved either in 100% ethanol for DNA extraction or in hot (steaming) 4% formalin for morphological examination. The avian definitive host was identified using the field guide of Howell & Webb (Reference Howell and Webb1995) and the American Ornithologists’ Union (1998) guidelines, and the nomenclature follows the Avibase database (http://avibase.bsc-eoc.org).
Amplification and sequencing of DNA
Two specimens were placed individually in tubes and digested overnight at 56°C in a solution containing 10 mm Tris-hydrochloride (pH 7.6), 20 mm sodium chloride, 100 mm Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2 EDTA) (pH 8.0), 1% sarkosyl and 0.1 mg/ml proteinase K. Following digestion, DNA was extracted from the supernatant using the DNAzol reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's instructions. The cytochrome cox 1 of mitochondrial DNA and D1–D3 domains of the LSU of nuclear ribosomal DNA were amplified using polymerase chain reaction (PCR). A fragment of cox 1 was amplified using the forward JB3 5′-TTTTTTGGGCATCCTGAGGTTTAT-3′ and reverse JB4.5 5′-TAAAGAAAGAACATAATGAAAATG-3′ primers (Bowles et al., Reference Bowles, Hope, Tiu, Liu and McManus1993).
Domains D1–D3 from LSU were amplified using forward primer 391, 5′-AGCGGAGGAAAAGAAACTAA-3′ (Nadler et al., Reference Nadler, D'Amelio, Fagerholm, Berland and Paggi2000), and reverse primer 536, 5′-CAGCTATCCTGAGGGAAAC-3′ (Garcia-Varela & Nadler, Reference Garci´a-Varela and Nadler2005). The amplification reactions (25 μl) consisted of 1 μl of each primer (10 μM), 2.5 μl of 10× buffer, 1.5 μl of 2 mm magnesium chloride, 0.5 μl of Ethylenediaminetetraacetic acid disodium salt dihydrate (dNTPs) (10 mm), 16.37 μl of water, 2 μl of genomic DNA and 1 U of Taq DNA polymerase (Platinum Taq, Invitrogen Corporation, São Paulo, Brazil). The PCR cycling conditions for amplification included denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 1 min, annealing at 48°C for cox 1 and 50°C for LSU for 1 min, and extension at 72°C for 1 min, with a final postamplification incubation at 72°C for 10 min. The sequencing reactions were performed using the initial primers for cox 1 and LSU plus two internal primers 503, 5′-CCTTGGTCCGTGTTTCAAGACG-3′ and 504, 5′-CGTCTTGAAACACGGACTAAGG-3′ (Garcia-Varela & Nadler, Reference Garci´a-Varela and Nadler2005) for LSU with ABI Big Dye (Applied Biosystems, Boston, MA, USA) terminator sequencing chemistry, and the reaction products were separated and detected using an ABI 3730 capillary DNA sequencer. Contigs were assembled, and base-calling differences were resolved using CodonCode Aligner 5.1.5 (CodonCode Corporation, Dedham, MA, USA). Sequences were deposited in the GenBank database (table 1).
Table 1. Specimens analysed in this study; host name, localities and GenBank accession numbers of each molecular marker.
Sequences in bold were generated in this study. B, bird; M, mammal.
Alignments and phylogenetic analyses
The new sequences were aligned using the software SeaView version 4 (Gouy et al., Reference Gouy, Guindon and Gascuel2010) and adjusted with the Mesquite program (Maddison & Maddison, Reference Maddison and Maddison2011). The LSU alignment included the new sequences plus 21 sequences from Lyperosomum spp. and seven sequences that were used as an outgroup (table 1). Two new cox 1 sequences were aligned with 17 other sequences of Lyperosomum spp., plus seven other sequences that were used as an outgroup (table 1). The phylogenetic analyses were performed using maximum likelihood (ML) and Bayesian inference (BI) methods. The ML analyses were carried out with RAxML version 7.0.4 (Silvestro & Michalak, Reference Silvestro and Michalak2011), and BI analyses were inferred with MrBayes version 3.2.7 (Ronquist et al., Reference Ronquist, Teslenko and Van der Mark2012) using the online interface Cyberinfrastructure for Phylogenetic Research (CIPRES) Science Gateway version 3.3 (Miller et al., Reference Miller, Pfeiffer and Schwartz2010). The best model was estimated with the Akaike information criterion using the jModel Test version 0.1.1 program (Posada, Reference Posada2008). The best model for each dataset was GTR + I + G for the LSU dataset and TPM3uf + I + G for the cox 1 dataset. ML analyses were inferred with models previously estimated for each molecular marker. To support each node, 10,000 bootstrap replicates were run. The BI analyses included Markov Chain Monte Carlo searches of two simultaneous runs for ten million generations, with sampling every 1000 generations, a heating parameter value of 0.2 and a ‘burn-in’ of 25%. Trees were drawn using FigureTree program version 1.3.1 (Rambaut, Reference Rambaut2012). The genetic divergence among taxa was estimated using uncorrected p-distances with the program MEGA version 6 (Tamura et al., Reference Tamura, Stecher, Peterson, Filipski and Kumar2013).
Morphological study
For taxonomic identification, seven specimens were stained with Mayer's paracarmine, dehydrated in a graded ethanol series, cleared with methyl salicylate and mounted on permanent slides with Canada balsam for deposition in the Colección Nacional de Helmintos (CNHE), Instituto de Biología, Universidad Nacional Autónoma de México (UNAM), Mexico City. Whole-mount specimens were examined using a Leica DM1000 LED (Leica Microsystems GmbH, Wetzlar, Germany) compound microscope. Measures are given in micrometres (μm). Illustrations of internal morphological features were produced using a drawing tube attached to a Leica MC120HD. For scanning electron microscopy (SEM), three specimens were dehydrated in a graded ethanol series, critical-point dried, sputter-coated with gold and examined with a Hitachi Stereoscan Model S-2469 N scanning electron microscope operating at 15 kV from the Instituto de Biología, UNAM.
Results
Lyperosomum cuauhxinqui sp. n.
Morphological description
Description (based on seven mounted adult specimens and three analysed by SEM). Measurements of holotype are provided in the description. Measurements of paratypes are provided in table 2.
Fig. 1. Lyperosomum cuauhxinqui sp. n. from Melanerpes aurifrons: (a) whole worm holotype, ventral view; (b) enlarged view of cirrus sac; (c) ovarian region showing relative position of ovary, seminal receptacle and Mehlis’ gland. Scale bars: (a) 500 μm; (b) 100 μm; (c) 50 μm.
Fig. 2. Scanning electron micrographs of the whole worm adults of Lyperosomum cuauhxinqui sp. n. from Melanerpes aurifrons: (a) whole worm; (b) oral sucker showing the arrangement of three pairs of papillae (arrows); (c) forebody. Scale bars: (a) 500 μm; (b) 100 μm; (c) 400 μm.
Table 2. Comparative measurements of Lyperosomum Looss, 1899 from the Americas.
Measurements in micrometres. Mean in brackets.
aEstimated from the published drawing.
Body elongate, slender, 3682 long. The maximum width in the ventral sucker region was 527 (figs 1a and 2a). Tegument thin with no spines, rough, with sensory conical papillae in the forebody (fig. 2c). Forebody short, 903. Forebody-to-body-length ratio 1:4. Suckers close to each other. Oral sucker terminal, round, 252 long, 218 wide, bearing three pairs of internal dome-like papillae (fig. 2a, b). Ventral sucker well developed, larger than the oral sucker, very muscular, situated at the end of the first third of the body, 431 long, 419 wide (figs 1a and 2c). Oral-to-ventral-sucker-length ratio 1:1.7. Oral-to-ventral-sucker-width ratio 1:1.9. (figs 1a and 2b). Prepharynx present, 15 long. Small pharynx 92 long, 119 wide. Oesophagus curved, 170 long. Caeca long, unequal in length, right caeca longer than the left (3111 and 2811, respectively), terminating in the posterior of the body (fig. 1a). Testes spherical, intercaecal, situated symmetrically just posterior to ventral sucker (fig. 1a), right testis 127 long, 94 wide, left testis 121 long, 91 wide. Cirrus sac small, oval, containing a sinuous seminal vesicle and cirrus, 136 long, 82 wide (fig. 1b). Cirrus sac situated posterior to the pharynx and anterior to the ventral sucker. Genital pore situated posterior to the pharynx. Ovary spherical, 119 long, 116 wide, distant from the testes, separated by numerous uterine coils (fig. 1c). Distance between testes and ovary, 481. Ovary submedial, located anterior to the middle of the body, dextral (n = 4) or sinestral (n = 3) position. Mehlis’ gland situated post-ovary, contiguous with the seminal receptacle. Seminal receptacle, oval, 81 long, 68 wide (fig. 1c). Laurer's canal not observed. Vitellarium conformed to numerous small follicles arranged in two lateral narrow rows, mostly in extracaecal fields. Vitelline fields asymmetrical, beginning at the posterior level of the testes, extending anteriorly to the end of the caeca. Right and left vitelline fields are 1547 and 1685 long, respectively. Vitelline-field-length-to-body-length ratio 1:2.1. Uterus convoluted, filling most of the hindbody, occupying the intertesticular region, dorsal to the ventral sucker. Mature eggs numerous, thick-walled, 20–32 long, 16–22 wide (n = 20). The excretory canals with thin walls originate on the excretory vesicle and extend to the anterior region at the level of the pharynx. Excretory vesicle I-shaped. Excretory pore terminal.
Taxonomic summary
Type host. Golden-fronted woodpecker, M. aurifrons (Wagler, 1829) (Aves: Piciformes: Picidae).
Type locality. Río Purificación (24°05′21.4″N, 99°09′54″W), Tamaulipas, México.
Site of infection. Intestine.
Date of collection. 17, February, 2016.
Type material. Holotype, CNHE 11245, and six paratypes, CNHE 11246.
Representative DNA sequences deposited. MT348379-80 (cox 1), MT340826 (LSU).
Etymology. The new species is named in reference to its definitive host, the golden-fronted woodpecker. The specific epithet derives from the Náhuatl language. Cuauhxinqui = woodpecker (‘carpintero’ in Spanish).
Note. All the measures presented in the description were recorded before the forebody of the type specimen was slightly damaged during the drawing process.
Remarks
The new species possesses features that are consistent with the diagnosis of the genus Lyperosomum: testes positioned closely to the ventral sucker, genital pore located anterior to the intestinal bifurcation, ovary positioned posterior to the testis, vitellarium forming two lateral bands of follicles extending anteriorly past the level of the ovary and a ventral sucker larger than the oral sucker (see Pojmańska, Reference Pojmańska, Bray, Gibson and Jones2008). In the Americas, four species of Lyperosomum have been found parasitizing birds and mammals. The new species can be differentiated from L. oswaldoi by a shorter body length (2263–3682 vs. 4300–10,650 in L. oswaldoi). The new species is also differentiated from L. byrdi by a greater body width (528–702 vs. 300–441 in L. byrdi). Lyperosomum cuauhxinqui sp. n. is differentiated from L. intermedium by a greater body width (528–702 vs. 335–420 in L. intermedium). In addition, the new species can be differentiated from L. oswaldoi, L. byrdi and L. intermedium by exhibiting symmetrical rather than oblique testes. Lyperosomum cuauhxinqui sp. n. is differentiated from the type species, L. petiolatum, by an oral sucker that is larger than that of L. petiolatum (213–315 × 219–285 vs. 130–200 × 130–200). Finally, the new species occurs in the intestine of its definitive host, whereas the other congeneric species occur in the liver, gallbladder, pancreas and bile ducts (see table 2).
Phylogenetic analysis
Nuclear marker
The LSU dataset included 1373 characters, and the best evolution model obtained was GTR + I + G. The alignment included 17 sequences representing six species of Lyperosomum and four other sequences belonging to Lyperosomum sp., in addition to sequences from seven other genera from Dicrocoeliidae that were used as an outgroup (see table 1). The phylogenetic tree inferred with the ML and BI methods suggests that the species of Lyperosomum, including the new species, form a monophyletic assemblage with strong bootstrap support (100%) and a high Bayesian posterior probability (1.0). The phylogenetic trees (fig. 3) showed that the species L. intermedium and Lyperosomum transcarpathicus (Bychovskaja-Pavlovskaja, Vysotzkaja & Kulakova, 1970), which are parasites isolated from mammals from the New World and Old World, respectively, formed two independent clades of Lyperosomum. The phylogenetic trees inferred with other species of parasites from birds were divided into two major clades. The first clade included two isolates of Lyperosomum clathratum (Deslongchamps, 1824) from the Czech Republic + Lyperosomum sp. (MK480326), in addition to three isolates of Lyperosomum turdia (Ku, 1938) from Poland and the Czech Republic + Lyperosomum sp. (MG560864) and a subclade that contained nine isolates of L. petiolatum from Poland and the Czech Republic. The second major clade included the species Skrjabinus kalmikensis (Skrjabin & Issaitschikow, 1927) (MK478495) plus two isolates of Lyperosomum sp. (MK496656, MK496657) from the Czech Republic. One subclade contained an isolate of Lyperosomum sarothrurae (Baer, 1959) from Africa and was sister to L. cuauhxinqui sp. n. plus Zonorchis alveyi (Martin & Gee, 1949) (MK480327), Zonorchis delectans (Travassos, 1944) (MK480329) and Zonorchis sp. (MK480328). All of these phylogenetic relationships received strong bootstrap support and showed very good Bayesian posterior probabilities (see fig. 3). The genetic divergence estimated among the species of Lyperosomum ranged from 2 to 6%, whereas the lowest genetic divergence found was 2%, between L. cuauhxinqui sp. n. and L. sarothrurae from Africa, and the greatest genetic divergence was 6%, between L. cuauhxinqui sp. n. and L. intermedium from the USA (table 3). Finally, the genetic divergence among isolates of L. clathratum, L. turdia and L. petiolatum ranged from 0 to 0.1% (table 3).
Fig. 3. Maximum likelihood (ML) tree and consensus Bayesian inference (BI) tree inferred from the large subunit from nuclear ribosomal DNA. Numbers near internal nodes show ML bootstrap clade frequencies and posterior probabilities (BI). Scale bar shows the number of substitutions per site.
Table 3. Pairwise nucleotide sequence comparisons among taxa for the LSU sequences (N = 1373 nt) (below the diagonal) and for the cox 1 sequences (N = 411 nt) (above the diagonal).
Mitochondrial marker
The cox 1 dataset included 411 characters with 26 terminals, and the best selected model was TPM3uf + I + G. This alignment included 16 specimens representing three species of Lyperosomum and a single sequence identified as Lyperosomum sp., plus six other genera from Dicrocoeliidae that were used as an outgroup. The phylogenetic tree inferred with the ML and BI methods suggested that the species of Lyperosomum form a monophyletic group (fig. 4) that is subdivided into two major clades. The first clade included an isolate of Lyperosomum sp. (MK445285) from the Czech Republic + S. kalmikensis (MK445286) and three isolates of L. clathratum from the Czech Republic. The second clade included two isolates of L. cuauhxinqui sp. n. from northern Mexico and was sister to another subclade formed by eight isolates of L. petiolatum plus five isolates of L. cf. turdia from the Czech Republic and Poland. The genetic divergence among the species of Lyperosomum ranged from 5.9 to 13.8%. The lowest genetic divergence value was 13.4%, between L. cuauhxinqui sp. n. and the isolates of L. petiolatum from the Czech Republic and Poland, and the greatest value was 17.3%, for L. clathratum from the Czech Republic. The genetic divergence among the isolates of L. clathratum, L. turdia and L. petiolatum ranged from 0 to 0.32%, and that between the two isolates of L. cuauhxinqui sp. n. was 0.2% (table 3).
Fig. 4. Maximum likelihood (ML) tree and consensus Bayesian inference (BI) tree inferred from the cox 1 of the mitochondrial DNA. Numbers near internal nodes show ML bootstrap clade frequencies and posterior probabilities (BI).
In summary, the phylogenetic analyses inferred with the LSU and cox 1 datasets are congruent in that L. cuauhxinqui sp. n. is nested within Lyperosomum. As a second result derived from the phylogenetic trees, the sequences identified as Z. alveyi (MK480327), Z. delectans (MK480329), Zonorchis sp. (MK480328) plus S. kalmikensus (MK478495 and MK445286) should be transferred to Lyperosomum.
Discussion
The genus Lyperosomum was erected to include four species, with Lyperosomum longicaudata (Rudolphi, 1809) as the type species. The species composition of the genus has been unsettled ever since, and, prior to the present study, it contained 33 valid species (see Hildebrand et al., Reference Hildebrand, Pulis and Tkach2015, Reference Hildebrand, Pyrka, Sitko, Jeżewski, Zaleśny, Tkach and Laskowski2019). Recently, Hildebrand et al. (Reference Hildebrand, Pyrka, Sitko, Jeżewski, Zaleśny, Tkach and Laskowski2019) performed a comprehensive study that included representative samples of Lyperosomum from the Americas and other regions of Europe and Africa; the authors of that study recognized several species on the basis of cox 1 and LSU sequences, which represents the starting point for further studies within the genus Lyperosomum. Therefore, the results obtained in the current study add new evidence that allows us to better understand the taxonomy and systematics of the genus Lyperosomum. Our phylogenetic trees inferred with the LSU dataset placed L. cuauhxinqui sp. n. in a subclade together with two other species of the genus Zonorchis (Z. alveyi (MK480327), Z. delectans (MK480329) and Zonorchis sp. (MK480328)) from the Americas (see fig. 3). The phylogenetic trees exhibited the same branching order as the phylogenetic tree inferred previously by Hildebrand et al. (Reference Hildebrand, Pyrka, Sitko, Jeżewski, Zaleśny, Tkach and Laskowski2019), with the exception of the branches belonging to the new species. The genus Zonorchis includes species from the Americas and differs morphologically from Lyperosomum in the position of the testes (oblique in Lyperosomum and symmetrical in Zonorchis). However, the inconsistency in the position of the testes has been the major reason for the taxonomic confusion between the species of the genera Lyperosomum and Zonorchis (see Denton & Byrd, Reference Denton and Byrd1951; Odening, Reference Odening1964; Hildebrand et al., Reference Hildebrand, Pyrka, Sitko, Jeżewski, Zaleśny, Tkach and Laskowski2019). For example, specimens of L. clathratum and L. petiolatum and the new species exhibit symmetrical testes, and based on this morphological trait, the three species should be placed in the genus Zonorchis. However, in the phylogenetic trees inferred with the LSU dataset, the species L. clathratum, L. petiolatum and L. cuauhxinqui sp. n. were nested within the genus Lyperosomum. Therefore, the position of the testes alone is not a suitable character for distinguishing between the two genera (see Hildebrand et al., Reference Hildebrand, Pyrka, Sitko, Jeżewski, Zaleśny, Tkach and Laskowski2019).
The four species of Lyperosomum found in the Americas associated with birds exhibit a wide spectrum of definitive hosts: L. petiolatum, L. oswaldoi and L. byrdi are associated with passerines of the families Corvidae, Mimidae and Passerellidae, respectively, whereas L. cuauhxinqui sp. n. is associated with the golden-fronted woodpecker (Picidae) distributed from Texas, USA, to Guatemala in Central America (Howell & Webb, Reference Howell and Webb1995). The genetic divergence estimated among the congeneric species of Lyperosomum ranged from 2 to 6% for LSU. The genetic divergence between L. cuauhxinqui sp. n. and its sister taxon species, such as Z. alveyi (MK480327), Z. delectans (MK480329) and Zonorchis sp. (MK480328), ranged from 0.7 to 1.6%; between two recognized species of Lyperosomum, L. turdia and L. petiolatum, the genetic divergence ranged from 0.4 to 0.56%; and between L. intermedium and L. transcarpathicus, it was 5.6%. These ranges of genetic divergence for LSU are wider than those previously described for congeneric species of Brachylecitum, ranging from 0.1 to 0.7% (see Hildebrand et al., Reference Hildebrand, Sitko, Zaleśny, Jeżewski and Laskowski2016). With respect to the cox 1 gene, the genetic divergence estimated among the five congeneric species of Lyperosomum ranged from 5.9 to 17.3%. The genetic divergence among L. cuauhxinqui sp. n. and its sister taxon species, such as L. turdia and L. petiolatum, ranged from 13.4 to 16.2%. The high level of genetic divergence among these species in the cox 1 gene confirms that the cox 1 gene evolves faster than LSU and that it is highly informative for distinguishing closely related species of digeneans.
Our phylogenetic trees inferred with LSU and cox 1 agree with the phylogenies previously inferred by Hildebrand et al. (Reference Hildebrand, Pyrka, Sitko, Jeżewski, Zaleśny, Tkach and Laskowski2019). The sequences identified as Z. alveyi, Z. delectans, Zonorchis sp. and S. kalmikensus were nested within Lyperosomum; therefore, these sequences should be considered to belong the genus Lyperosomum. It is imperative to review the taxonomy of the genus Lyperosomum by using a combination of ecological, morphological and molecular characteristics, as suggested previously by Hildebrand et al. (Reference Hildebrand, Pyrka, Sitko, Jeżewski, Zaleśny, Tkach and Laskowski2019), with the aim of building a more robust classification of the genus that allows us to better understand the evolution of this group of digeneans.
Acknowledgements
We are grateful to Carlos Daniel Pinacho and Alejandra López Jiménez for their help during field work. We also thank Berenit Mendoza for her help with the use of the SEM unit, Luis García Prieto for providing material from the CNHE and Laura Márquez and Nelly López Ortiz from LaNabio for their help during the sequencing of the DNA fragments.
Financial support
This research was supported by grants from the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT-UNAM) IN207219.
Conflicts of interest
None.
Ethical standards
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.
