Introduction
Bats (Mammalia: Chiroptera) are the second-largest group of mammals on earth (surpassed only by Rodentia), with approximately 1460 species recorded worldwide (Burgin et al., Reference Burgin, Colella, Kahn and Upham2018). One of the areas with the greatest diversity of bats is the Neotropical Region; particularly for Mexico, bats constitute about 15% of the total mammals distributed in the country (Ceballos et al., Reference Ceballos, Arroyo-Cabrales, Medellín and Domínguez-Castellanos2005).
This group is an excellent environment to study symbiotic (host-parasite) interactions because they host many distinct kinds of eukaryotic macrosymbionts with parasitic strategies, such as helminths (Dick & Patterson, Reference Dick and Patterson2006; Morand et al., Reference Morand, Krasnov and Poulin2007; Dittmar et al., Reference Dittmar, Morse, Dick, Patterson, Morand, Krasnov and Littlewood2015; Jiménez et al., Reference Jiménez, Caspeta-Mandujano, Ramírez-Chávez, Ramírez-Díaz, Juárez-Urbina, Peralta-Rodríguez and Guerrero2017). The helminths linked to this group have been the subject of irregular, local, and asymmetrical research from a helminthological perspective for Mexico and Latin America (Caspeta et al., Reference Caspeta-Mandujano, Peralta-Rodríguez, Ramírez-Chávez, Ramírez-Díaz, Tapia-Osorio, Juárez-Urbina, Guerrero-Enríquez, Galindo-García, Martínez-Rodríguez, Ojeda and Juárez-González2017).
According to data from the Colección Nacional de Helmintos (UNAM, 2024, Data not published), there are 58 species of helminths in Mexico. These comprise 26 digeneans, five cestodes, and 27 nematodes. These numbers come from just 29 of the country’s 140 species of bats that have been recorded. From this perspective, the group’s knowledge has advanced by only 20.71%. Seven of the eight families of bats distributed in Mexico have reported helminth species; the most represented is Phyllostomidae, with 11 species examined, and the least studied is Emballonuridae, with only one species. Because of this difference, more in-depth studies are needed to finish the helminthological record of bats, especially in places with a great deal endemism and/or human-caused disturbance (Jiménez et al., Reference Jiménez, Caspeta-Mandujano, Ramírez-Chávez, Ramírez-Díaz, Juárez-Urbina, Peralta-Rodríguez and Guerrero2017). As part of a larger project to analyze the relationship between helminth richness and microbial composition in Neotropical bats, we collected a species of digenean belonging to the superfamily Microphalloidea. The aim of the present work is to describe a new genus and species of this superfamily, a parasite of two species of chiropterans of the genus Peropteryx (Emballonuridae) from southeast Mexico, and to determine its phylogenetic position within this superfamily based on morphological and molecular characteristics.
Material and Methods
Collection of hosts and helminths
During December 2021, three males and three females of Peropteryx kappleri Peters, 1867 collected in Chajul, Chiapas, and one male specimen of the Lesser dog-like bat Peropteryx macrotis Wagner, 1863, collected in August 2022, from Grutas las Sartenejas, Tekax, Yucatán, were examined for trematodes (Figure 1). The sampling followed the protocols established by the American Society of Mammalogists (Sikes et al., Reference Sikes, Thompson and Bryan2019) and the Food and Agriculture Organization (El Masry et al., 2020), under the scientific collector’s permit DGVS/04214 /19 issued to ORC by the Secretaría del Medio Ambiente y Recursos Naturales de México. Each bat was kept individually in cotton bags and transferred to the laboratory. Its taxonomic identification was made with the help of a field guide (Medellín et al., Reference Medellín, Arita and Sánchez2008). The hosts were euthanized via inhaled isoflurane overdose, and an examination was performed on all internal organs to detect trematodes in situ. For morphological analysis, worms were fixed in hot 4% formalin and stored in 70% ethanol; for molecular analysis, they were preserved in 96% ethanol. Infection parameters follow Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997).
Map of Mexico showing the sampled sites for chiropterans.
Morphological analysis
For permanent mounts, the material was stained with Mayer’s paracarmine and mounted in Canada balsam. All measurements are given in micrometres (μm) unless stated otherwise. They are shown as the mean followed by the standard deviation (± SD), and the range and number of specimens measured (n) in parentheses. The drawings were made with the aid of a drawing tube. The individuals studied under scanning electron microscopy (SEM) were dehydrated in alcohol graduated to 100% ethanol. Subsequently, they were critically dried with CO2 and mounted to be coated with a mixture of gold and palladium. The observation of samples was made under a Hitachi S2460N microscope in the Laboratory of Microscopy and Photography of Biodiversity I, of the Institute of Biology, Universidad Nacional Autónoma de México (IBUNAM), Mexico City. Type specimens were deposited in the Colección Nacional de Helmintos, Instituto de Biología, IBUNAM.
DNA extraction and 28S rRNA gene amplification and sequencing
Total genomic DNA from eight adult worms was extracted with the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) modifying slightly the manufacturer’s protocol: To concentrate DNA, this was eluted with 300 μL of AE buffer and precipitated with absolute ethanol, 0.1 volumes of 3M sodium acetate, and 2 μL of glycoblue. DNA was re-suspended in 30 μL of molecular grade water and stored at –20 °C until PCR amplification (Gaona et al., Reference Gaona, Cerqueda-García, Moya, Neri-Barrios and Falcón2020). An approximately 1467-bp-long region at the 5′ end of the nuclear of the 28S ribosomal RNA gene region (D1-D3) was amplified using primers forward 391 5′ AGCGGAGGAAAAAGAAACTAA-3′ (Nadler & Hudspeth, Reference Nadler and Hudspeth1998) and reverse 536 5′-CAGCTATCCTGAGGGAAAC-3′ (Stock et al., Reference Stock, Campbell and Nadler2001). Each PCR reaction was made using DNA MyTaq Mix polymerase (Bioline); annealing temperature during the thermal cycling was 50 °C. PCR products were purified with ExoSAP-IT (Applied Biosystems, Inc.), following the manufacturer’s instructions. Sequencing reactions were performed with the two amplification primers and the internal primers 503 and 504 (see in Mendoza-Garfías et al., Reference Mendoza-Garfías, García-Teh, Caspeta-Mandujano, Vidal-Martínez and Hernández-Mena2022) using BigDye Terminator v3.1 Cycle sequencing Kit (Applied Biosystems, Inc.), and products were purified by filtering through hydrated Sephadex (GE Healthcare) columns. DNA Sequencer ABI PRISM 3730 (Applied Biosystems, Carlsbad, CA) was used to generate DNA sequences at the Laboratorio de Secuenciación Genómica de la Biodiversidad, IBUNAM. The consensus DNA sequences for each specimen were assembled using Geneious Pro 4.8.4 software (Biomatters; http://www.geneious.com/). DNA sequences were subjected to a BLAST search using the NCBI website (http://www.ncbi.nlm.nih.gov) to discard possible contamination or amplification of host DNA. All DNA sequences generated in the present study were submitted to GenBank under accession numbers: PQ069219-PQ069227.
DNA sequence alignment and phylogenetic analyses
A data set for the 28S rRNA gene was built in Mesquite 3.62 (http://www.mesquiteproject.org/), with the new sequences generated in this study and deposited in Genbank. Macroderoides spiniferus (EU850400) was used to give a root to the trees obtained from the Microphalloidea superfamily. DNA sequences were aligned using MAFFT (Katoh et al., Reference Katoh, Rozewicki and Yamada2018) through the web site https://mafft.cbrc.jp/alignment/server/index.html with the default settings for opening and gap extension. The substitution models and parameters were selected in jModelTest v.2.1.6 (Posada, Reference Posada2008), based on the Akaike Information Criterion (Akaike, Reference Akaike, Petrov and Csaki1973). Phylogenetic inference was performed with the data set of the 28S rRNA gene, following two different approaches. The tree obtained from Maximum Likelihood (ML) was implemented using RAxMLGUI v 1.3 (Stamatakis, Reference Stamatakis2006); to obtain node support, a bootstrap analysis (Bt) was performed with 1,000 replicates using the GTR+I+G nucleotide evolution model. Bayesian inference (BI) was performed in Mr. Bayes 3.2. (Huelsenbeck & Ronquist, Reference Huelsenbeck, Fredrik, Nielsen and Bollback2001) using four independent Markov chain Monte Carlo runs, each comprising four chains (one cold, three heated), and a random starting tree; each chain was run for 20,000,000 generations, with 10% discarded as burn-in. Trees were sampled every 1,000 generations. After the burn-in, the remaining samples from the four runs were combined to estimate the topology, branch lengths, and posterior probabilities (PP). Finally, convergence of the Markov chain Monte Carlo runs was assessed by visual inspection of trace plots for all parameters using Tracer v. 1.7 (Rambaut et al., Reference Rambaut, Drummond, Xie, Baele and Suchard2018). Both RaxML and MrBayes were used online on the CIPRES Science Gateway (Miller et al., Reference Miller, Pfeiffer, Schwartz, Miller, Pfeiffer and Schwartz2010). The phylogenetic trees obtained from both analyses were visualized in FigTree v. 1.4.2.
Results
Morphological descriptions
Superfamily: Microphalloidea Ward, 1901
Family: Incertae sedis
Diagnosis. Digenea, Microphalloidea. Body elongated, arrow-shaped forebody, narrowing posteriorly or to level of ventral sucker. Body completely covered by spines,. Oral sucker spherical and terminal, larger than ventral sucker. Preequatorial and intercecal ventral sucker. Prepharynx absent. Oval pharynx. Relatively short esophagus; cecal bifurcation anterior to vitelline follicles, cirrus sac, and ventral sucker. Intestinal caeca end at level of the posterior end of ovary. Testes symmetrical, entire, separated, post-equatorial. Proximal part of cirrus-sac anterior; cirrus-sac oriented posteriorly from this point, very long, forming several loops, lateral to ventral sucker (dextral), containing elongate internal seminal vesicle, pars prostatic (bulb shaped), and wide muscular ejaculatory duct. Genital pore ventral, median, posterior to ventral sucker. Ovary lateral (sinistral), slightly posterior to ventral sucker. Ootype overlapping ovary. Seminal receptacle lateral (sinistral), pre-ovarian, slightly anterior to ventral sucker. Laurer’s canal not observed. Mehlis’ gland posterior to ventral sucker. Vitelline follicles confluent in forebody, beginning posteriorly to intestinal bifurcation, extending at anterior end of ovary. Vitelline reservoir ventral to Mehlis’ gland frequently posterior to ventral sucker. Uterus coiled, occupying most of hind-body; metraterm not observed. Eggs operculated, not embryonated. Excretory vesicle I-shaped, reaching posterior margin of posterior testis. Excretory pore terminal. Parasites of bats in Neotropical region.
Type and single species: Sagittatrema zutzi n. sp.
Etymology: The generic name comes from the characteristic arrow-shaped morphology of the body of this digenea.
Remarks: The new genus clearly pertains to Microphalloidea following Bray (Reference Bray, Bray, Gibson and Jones2008). However, it cannot be included in one of the 17 families contained in this superfamily by the position of genital pore in hindbody, in the midline of body, posterior to ventral sucker. Collyryclum, another microphallid genus also has a genital pore posterior to the ventral sucker; notwithstanding, their genital ducts open on a small nipple. In addition, the sagittiform body of the new genus is an exclusive trait within the superfamily. Furthermore, it can be distinguished from the members of the seven phylogenetically closest families (Figure 4) by the following morphological features: (1) distribution of the vitelline glands: in the genera of Collyriclidae exceed the cecal bifurcation and those of Prosthogonimidae are arranged in lateral fields not confluent, that reach the posterior end, whereas in Sagittatrema, they constitute a single group in the anterior part of the body, confluent at the level of the cecal bifurcation. (2) The extension of the ceca: genera of Colyrichlidae and Stomylotrematidae, they reach the posterior end of the body; in the members of Microphallidae and Lecithodendriidae, they generally do not extend beyond the ventral sucker and in the genera of the families Phaneropsolidae, Pleurogenidae, and Prosthogonimidae, their extent is variable compared to those presented by Sagittatrema, where ceca reach into the hindbody but not as far testes. Finally, (3) by the site of infection: Sagittatrema parasitizes the gallbladder and intestine, whereas the genera of five of the seven families parasitize only the intestine (Microphallidae, Phaneropsolidae, Lecithodendriidae, Prosthogonimidae, and Stomylotrematidae) or are encyst in the intestinal wall (Collyriclidae) or in the hepatic ducts (Pleurogenidae) (Table 1).
Main morphological characters of families of Microphalloidea* included in the phylogenetic analyses; traits of the new genus are highlighted
* Based on Bray et al. (Reference Bray, Bray, Gibson and Jones2008).
Sagittatrema zutzi sp. nov. (Figures 2, 3,4)
Sagittatrema zutzi n. sp. (entire adult worm, ventral view) from Peropteryx kappleri. a) line drawings, ventral view, and b) optical microscopy photograph. Scale bars: a) and b) 500 μm.
Detail of male and female reproductive systems. Scale bar: 8 μm.
Scanning electron micrographs of Sagittatrema zutzi n. sp., from Peropteryx kappleri. a) adult, ventral view, b) anterior region, oral sucker, c) spines, equatorial region, d) ventral sucker, and e) excretory pore. Scale bars: a) 500 μm b) 50 μm; c) 20 μm; d) 50 μm, and e) 20 μm.
Description based on 11 specimens mounted in Canada balsam and 2 studied under SEM. Body large, arrow-shaped forebody, narrowing posteriorly at level of ventral sucker, 1.49 mm ± 0.40 (1.07–2.12 mm; n = 11) long, 0.61 ± 0.14 mm (0.40–0.85 mm; n = 11) maximum width. Body completely covered by spines. Length of spines at anterior end of body 8.75 ± 0.89 (5.5–10; n = 20); at mid-body 9.43 ± 1.11 (7.36–11.6; n = 20); at posterior end 8.77 ± 0.98 (7.05–10.3; n = 17). Oral sucker terminal, muscular, oval, 0.16 ± 0.03 (0.11–0.21, n = 11) long by 0.15 ± 0.02 (0.11–0.18; n = 11) wide. Ventral sucker muscular, rounded 0.13 ± 0.02 (0.09–0.17; n = 11) long, 0.14 ± 0.02 (0.10–0.18; n = 11) wide, at midline of body, pre-equatorial. Ratio oral sucker length/ventral sucker length 1:1.18 (1.06-1.27; n = 8). Prepharynx absent. Pharynx muscular, oval-shaped, 0.06 ± 0.007 (0.04–0.07; n = 11) long, 0.9 ± 0.006 (0.05–0.07, n = 11) wide. Esophagus 0.085 ± 0.27 (0.055–0.12; n = 5) long. Intestine bifurcates anterior to vitelline follicles. Intestinal caeca thin, ending between ovary and testes. Testes two, oblong, symmetrical, separated, at beginning of last third of body: right testis 0.23 ± 0.05 (0.18–0.33, n = 9) long, 0.16 ± 0.03 (0.13–0.21, n = 9) wide; left testis 0.22 ± 0.03 (0.18–0.29, n = 9) long, 0.17 ± 0.04 (0.09–0.23, n = 9) wide. Cirrus sac oriented longitudinally, very long, forming several loops, passes dextrally to ventral sucker, containing elongate internal seminal vesicle, bulb-shaped pars prostatica, and wide muscular ejaculatory duct. Genital pore opening on ventral surface, posterior to ventral sucker, on median line of body. Ovary ellipsoidal, posterior to ventral sucker, pre-testicular, 0.14 ± 0.03 (0.10–0.17, n = 9) long, 0.10 ± 0.02 (0.07–0.14, n = 9) wide. Ootype cylindrical, overlapping ovary dextrally, 0.082 ± 0.002 (0.0115–0.055, n = 6) long, 0.041 ± 0.010 (0.024–0.052, n = 6). Seminal receptacle sinistral-lateral, pre-ovarian, slightly anterior to ventral sucker. Laurer’s canal not observed. Mehlis’ gland posterior to ventral sucker. Vitelline follicles confluent posteriorly to intestinal bifurcation, extending to level of posterior margin of ventral sucker. Vitelline reservoir ventral to Mehlis’ gland, surrounding posterior area of ventral sucker. Uterus long, occupying almost all hindbody. Eggs ovate, numerous, operculated, not embryonated, 0.024 ± 0.011 (0.019–0.055, n = 100) long, 0.0110 ± 0.0007 (0.009–0.0119, n = 100) wide. Excretory vesicle I-shaped.
ZooBank Life Science Identifier: urn:lsid:zoobank.org:act:DBCDAC35-B1A0-4F83-B082-E4312E591693
Taxonomic summary
Type Host: Peropteryx kappleri Peters, 1867, Greater dog-like bat (Chiroptera: Emballonuridae).
Site of infection: Gallbladder and intestine.
Type locality: Boca de Chajul (16°6’58" N; 90°55’25"W), Chiapas, Mexico.
Other host: Peropteryx macrotis Wagner, 1863, lesser dog-like bat (Chiroptera: Emballonuridae).
Other locality: Grutas las Sartenejas, Tekax, Yucatán (20°12’8.3" N; 89°17’16.1"W).
Infection parameters: Chiapas: prevalence 4/6 (66%); mean abundance 4.16; mean intensity 6.25; intensity range: 1-11. Yucatán: 1/1 (100%) and intensity 2/1(2).
Specimens deposited: Holotype (CNHE 12854); 10 paratypes from P. kappleri (CNHE 12855) and 1 paratype from P. macrotis (CNHE 12856).
Etymology: The species name refers to the host group of the new species. Zutzi means bat in Mayan, the language of the region where this species was found.
Remarks
Two genera belonging to different families within Microphalloidea are the most similar to the new genus described here: Parabascus (Pleurogenidae see Lotz & Font, Reference Lotz, Font, Bray, Gibson and Jones2008) by the distribution of vitelline follicles mainly in the hindbody (Kirillova et al., Reference Kirillova, Shchenkov, Kirillov and Ruchin2022), and the monospecific genus Collyriclum (Collyriclidae) by the position of the genital pore almost in the mid-line of the body (Blair & Barton, Reference Blair, Barton, Bray, Gibson and Jones2008). Sagittatrema sutzi n. sp. can be differentiated from the only species of Parabascus distributed in Mexico (Parabascus yucatanensis [Stunkard, Reference Stunkard1938] Odening, 1969]) because the ovary in S. sutzi n. sp. is dextral to the ventral sucker, whereas in P. yucatanensis it is sinistral to this structure. In addition, in the species of Stunkard, the intestinal caeca reach the posterior end of the testes, whereas in S. sutzi n. sp., they only overpass the ventral sucker. Other differences between P. yucatanensis and the new species are the oval-shaped body and the lateral genital pore with separated sexes (Stunkard, Reference Stunkard1938), versus the body-arrow-shaped and common genital pore located in the mid-body line, respectively. On the other hand, Collyriclum faba (Bremser in Schmalz, 1831) Kossack, 1911, has separate genital pores, almost adjacent to one another on a small nipple (traits absent in the new species), sets of spaced spines, lacks a ventral sucker, and has clusters of non-confluent vitelline follicles, inhabiting subcutaneous cysts (Blair & Barton, Reference Blair, Barton, Bray, Gibson and Jones2008), whereas in S. sutzi n. sp., the body is covered completely with spines, has a ventral sucker, its vitelline follicles are confluent, and it infects the gallbladder and intestine.
Phylogenetic analyses
The analysed data set, based on 61 taxa from seven families of Microphalloidea (including nine sequences of Sagittatrema zutzi n. sp.) had a length of 1325 pair of bases (bp). The nucleotide substitution model that best fit this set was GTR+GAMMA+I. The nucleotide frequencies were A = 0.21, C = 0.22, G = 0.32 and T = 0.24. The trees obtained from ML and BI practically had the same topology and showed the same phylogenetic relationships, although in the node support, the Bt values were generally lower than the posterior probabilities. The ML tree had a value of 12007.1461. Both phylogenies recovered S. zutzi as a monophyletic group, with Bt = 100; PP = 1, support values. The Microphalloidea families were distributed into seven clades (Figure 5). However, the specimens sequenced of this study nested within the clade formed by the families: Collyriclidae Ward, 1917, Phaneropsolidae Mehra, 1935, Prosthogonimidae Lühe, 1909 and Pleurogenidae Looss, 1899 (Bt = 96; PP = 0.99). It is important to emphasize that in this tree, both Pleurogenidae (sensu Tkach et al. Reference Tkach, Chermak, Patitucci, Greiman, Thi Binh and Olson2023) and Phaneropsolidae are not monophyletic, because Collyriclum (Bremser in Schmalz, 1831) (Collyriclidae) was nested within Pleurogenidae, and Phaneropsolus Looss, 1899 and Limatulum Macy, 1931 (Phaneropsolidae) are in separated groups. More specifically, the new genus grouped with Prosthogonimidae and Collyriclidae + Pleurogenidae. The Pleurogenidae (sensu Tkach et al., Reference Tkach, Chermak, Patitucci, Greiman, Thi Binh and Olson2023) + Collyriclidae clade was the sister group to the Sagittatrema + Limatulum clade (Bt = 96; PP = 0.99). Finally, Sagittatrema and Limatulum clustered together with high bootstrap and posterior probability values (Bt = 93; PP = 0.99).
Phylogenetic hypothesis of the superfamily Microphalloidea. Trees inferred with Maximum Likelihood (ML) and consensus Bayesian Inference (BI), based on 28s rDNA gene sequences. Numbers near internal nodes show ML bootstrap percentage (BP) values and Bayesian posterior probabilities (BPP). The clade highlighted in bold indicates the position of the new genus and species studied in this work. Scale bars represent the branch length.
Discussion
Sagittarema sutzi n. sp., represents the first record for the Peropteryx bat genus in the Mexican territory; previously, only digenean flatworms of the genera Castroia, Limatulum, Ochoterenatrema and Urotrema had been recorded in Colombia and Brazil for these bats (Santos & Gibson, Reference Santos and Gibson2015). Morphologically, the new genus distinguishes itself from other members of the superfamily Microphalloidea with a sagittiform body, an excretory pore in the midline that does not protrude into a nipple and is posterior to the ventral sucker, and a very large cirrus sac that surrounds almost the entire ventral sucker. Additionally, the phylogenetic position of Sagittatrema using the 28s rRNA ribosomal gene places it in a monophyletic clade well supported in the two analyses performed (Bt =100; PP = 1). In our study, we obtained phylogenetic relationships within Microphalloidea consistent with previous proposals (Pérez-Ponce de León & Hernández-Mena, Reference Pérez-Ponce de León and Hernández-Mena2019; Tkach et al., Reference Tkach, Littlewood, Olson, Kinsella and Swiderski2003; Reference Tkach, Greiman, Pulis, Brooks and Bonilla2019; Reference Tkach, Chermak, Patitucci, Greiman, Thi Binh and Olson2023). However, in this case of the new genus, although it is related to Pleurogenidae (sensu Tkach et al., Reference Tkach, Chermak, Patitucci, Greiman, Thi Binh and Olson2023), it is not clear that it is part of this family. Based on these results, we considered that Sagittatrema n. gen. is not part of Pleurogenidae for several reasons: (1) Pleurogenidae is not monophyletic because of the inclusion of Collyriclum in our analysis as a sister group to the pleurogenid Loxogenes, suggesting that the position of Collyriclidae and the relationships between the internal clades of Pleurogenidae are not yet resolved; this coincides with previous phylogenetic hypotheses (Shchenkov et al., Reference Shchenkov, Denisova, Kremnev and Dobrovolskij2020; Sokolov et al., Reference Sokolov, Shchenkov, Kalmykov and Smirnova2020; Kirillova et al., Reference Kirillova, Shchenkov, Kirillov and Ruchin2022; Vlasenkov et al., Reference Vlasenkov, Kalmykov and Sokolov2023); however, we agree with Vlasenkov et al., (Reference Vlasenkov, Kalmykov and Sokolov2023) who pointed out that elimination of Collyriclidae and the inclusion of Collyriclum in Pleurogeidade are not possible at the moment because of the lack of morphological evidence to support it; this is also due to the constant eliminations and inclusions of genera in this family and (2) the nesting of Sagittatrema n. gen., with Limatulum making Phaneropsolidae apparently paraphyletic, which is consistent with the previous phylogenetic analysis of Moguel-Chin et al., (Reference Moguel-Chin, Panti-May, García-García and Hernández-Mena2024). Furthermore, Limatulum is a genus whose morphology does not exhibit all the diagnostic characters of Pleurogenidae. According to a more detailed review, the clade comprising Sagittatrema + Limatulum suggests the eventual creation of a new family within Microphalloidea, a possibility currently under analysis. However, the establishment of a new family requires the acquisition of a larger number of additional morphological and molecular data, so in the present work we prefer to adopt a more conservative approach and maintain Sagittatrema as a genus incertae sedis.
As mentioned by Tkach et al., (Reference Tkach, Greiman, Pulis, Brooks and Bonilla2019), this publication, along with other recent ones that preceded it on the phylogenetic systematics of Microphalloidea, “it is yet another step toward the re-organization and improvement of the systematics of this highly diverse, derived lineage of digeneans”.
Conflict interest declaration
The authors declare that they have no conflict of interest.
Acknowledgements
We are grateful to the fieldwork collaborators Karen Martínez, Shamayim M. Sánchez, and the Lombera family in Boca de Chajul, Chiapas. We thank Marco Torres Castro for his support in the fieldwork in Yucatán. To the collaborators of the Instituto de Ecología, UNAM: Osiris Gaona, Arit León Lorenzana and Alfredo Yanez Montalvo for technical support. Laura Márquez Valdelamar and Nelly María López Ortiz for the genomic sequencing process, LANABIO and Berenit Mendoza Garfias form LABNABIO for SEM micrographs. Georgina Ortega Leite provided important bibliographic references. Maribel Arenas Navarro and Alejandro Zaldivar Gómez for the creation of the map of the sampled localities. The first author thanks the Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCyT) for the scholarship awarded (number 583329) to carry out postgraduate studies in the Posgrado en Ciencias Biológicas-UNAM.
Financial support
This project was financed by the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica IN218020 (PAPIIT-UNAM).
Ethical standard
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.
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