A new cryptic species of Echinostoma (Trematoda: Echinostomatidae) closely related to Echinostoma paraensei found in Brazil

Abstract Abstract Echinostoma paraensei, described in Brazil at the end of the 1960s and used as a biological model for a range of studies, belongs to the ‘revolutum’ complex of Echinostoma comprising species with 37 collar spines. However, molecular data are available only for a few isolates maintained under laboratory conditions, with molecular prospecting based on specimens originating from naturally infected hosts virtually lacking. The present study describes Echinostoma maldonadoi Valadão, Alves & Pinto n. sp., a species cryptically related to E. paraensei found in Brazil. Larval stages (cercariae, metacercariae and rediae) of the new species were found in the physid snail Stenophysa marmorata in the State of Minas Gerais, Brazil, the same geographical area where E. paraensei was originally described. Adult parasites obtained experimentally in Meriones unguiculatus were used for morphological (optical microscopy) and molecular [28S, internal transcribed spacer (ITS), nad1 and cox1] characterization. The morphology of larval and adult parasites (most notable the small-sized dorsal spines in the head collar), associated with low (0–0.1%) molecular divergence for 28S gene or ITS region, and only moderate divergence for the mitochondrial cox1 gene (3.83%), might suggest that the newly collected specimens should be assigned to E. paraensei. However, higher genetic divergence (6.16–6.39%) was found in the mitochondrial nad1, revealing that it is a genetically distinct, cryptic lineage. In the most informative phylogenetic reconstruction, based on nad1, E. maldonadoi n. sp. exhibited a strongly supported sister relationship with E. paraensei, which may indicate a very recent speciation event giving rise to these 2 species.


Introduction
Species of the genus Echinostoma Rudolphi, 1809 are digenetic trematodes found, in the adult stage, in the intestine of birds and mammals, including humans (Fried and Graczyk, 2000;Toledo et al., 2009;Chai et al., 2020). More than 120 nominal species have been described worldwide making it the most diverse genus in the Echinostomatidae (Kostadinova and Gibson, 2000), but the pronounced morphological uniformity among species, along with other historical issues, has led to a complex taxonomic scenario. This is particularly true for the 'revolutum' group, formed by species of Echinostoma possessing 37 collar spines. About 60 nominal species were described worldwide (Chai et al., 2020), but the validity of most of them still requires confirmation, given that this group has been subjected to intense debate and constant taxonomic rearrangements over time (Georgieva et al., 2013(Georgieva et al., , 2014Faltýnková et al., 2015).
In the last decades, the use of molecular tools has revealed the existence of numerous independent evolutionary lineages of Echinostoma, usually referred to as cryptic species, but also has helped to unravel the correct identity of previously known taxa, some of them described as new (Detwiler et al., 2010;Georgieva et al., 2013Georgieva et al., , 2014Georgieva et al., , 2017Faltýnková et al., 2015;Izrailskaia et al., 2021;Valadão et al., 2022). These recent advances have contributed to a better knowledge of the species composition and the distribution of these parasites and have generated evidence on their biogeography, evolution and radiation. However, such integrative taxonomic studies involving Echinostoma spp. are disproportionately carried out in Asia, Europe and North America (Detwiler et al., 2010;Georgieva et al., 2014;Chai et al., 2021). In fact, in South America, molecular studies involving species of Echinostoma are scarce (Maldonado et al., 2001a;Valadão et al., 2022) and do not reflect the diversity of species known in this subcontinent (more than 30 species, 12 bearing 37 collar spines) (Travassos et al., 1969;Fernandes et al., 2015;Lunaschi et al., 2018;Valadão et al., 2022).
Despite this tremendous scientific knowledge accumulated over decades about E. paraensei, studies on the molecular characterization of the species are scarce. Considering genetic markers, commonly used in taxonomic studies of trematodes, few sequences generated for E. paraensei are available in the public genetic sequence databases. They correspond to the complete mitochondrial genome of the isolate maintained under laboratory conditions in the USA (KT008005 -Adema et al., direct submission in GenBank®, unpublished), some internal transcribed spacer (ITS) sequences generated for the Brazilian isolate from N. squamipes (Maldonado et al., 2001a) and nad1 sequences obtained for larval stages found in Australia and for the isolate originated from Brazil and maintained in the USA (Morgan and Blair, 1998a).
In the present study, we provide data obtained during experimental, morphological and molecular studies of a 37-collar-spined echinostome found in Stenophysa marmorata (Guilding, 1828) from Brazil. Despite the morphological data of the developmental stages and sequences of nuclear genetic markers (28S and ITS) suggested that the parasite is indistinguishable from E. paraensei, mitochondrial data (nad1 gene) pointed to a genetically distinct lineage, herein described as a new species of Echinostoma.

Sampling and experimental infection
The isolate of the parasite evaluated in this study was obtained from a specimen of S. marmorata collected in the municipality of Alvorada de Minas, Minas Gerais, Brazil, in February 2019 during a malacological survey focused on the epidemiology of schistosomiasis. Details of malacological techniques and preliminary evaluation of infection with larval trematodes are as described in Coelho et al. (2021Coelho et al. ( , 2022. The infected snail was sent to the laboratory for further parasite identification. A new photostimulation test was carried out for the recovery of cercariae. The infected mollusc died on the following day and was dissected for retrieval of rediae and metacercariae. This fact precludes us from providing detailed illustrations of larval stages. For the recovery of adult parasites, part of the collected metacercariae were used to infect a laboratory-reared jird, Meriones unguiculatus (Milne-Edwards, 1867). The rodent was orally inoculated with approximately 60 metacercariae. The infected animal was submitted to an immunosuppression protocol through subcutaneous inoculation of dexamethasone disodium phosphate (Decadron, Ache, Brazil), 25 mg kg −1 , daily until the end of the experiment. The success of infection was verified by fecal examination through the spontaneous sedimentation method (Lutz, 1919) from 7 days post-infection (dpi) onwards. The animal was kept in the laboratory with water and food provided ad libitum until the euthanasia carried out at 15 dpi. The intestine was transferred to Petri dishes containing physiological saline (0.85% NaCl) and examined under a stereomicroscope for the presence of adult or juvenile parasites.

Morphological study
Samples of the emerged cercariae were stained with vital stains (0.05% neutral red or Nile blue sulphate) and mounted in temporary preparations for examination under a light microscope. Larvae were also killed in hot water and fixed in 10% formalin for morphometric analysis. Rediae and metacercariae were studied live in temporary preparations and then also fixed in 10% formalin. Adult parasites collected from the experimentally infected jird were individually compressed between glass slides and fixed in 10% formalin. Later, they were stained with alum acetocarmine, dehydrated in an increasing series of ethanol, clarified in beechwood creosote and mounted on permanent slides with Canada balsam. For the study of the cephalic collar, some specimens had their anterior extremity sectioned with the aid of a scalpel blade, transferred to a glass slide and clarified with Amman's lactophenol. The preparations were examined under the microscope for determination of the number, size, shape and arrangement of collar spines, according to . Whole adult parasites were photographed using a digital camera Samsung® ES70 (Samsung, Brazil), coupled to a stereomicroscope. Details of structures of adults and the larval stages were photographed with a Leica ICC50 HD digital camera, coupled to a Leica DM500 microscope. Captured images were analysed with Leica Application Suite software (LAZ EZ), version 2.0 (Leica Microsystems, Germany). The drawings were made with the aid of a camera lucida connected to an Olympus BH-2 microscope (Olympus, Japan). The morphometric analyses were carried out using a micrometre eyepiece. The abbreviations used in the morphological descriptions are in accordance with the most recent descriptions of Echinostoma (Kostadinova, 2005, Faltýnková et al., 2015Georgieva et al., 2017). In the case of dorsal spines (DLSL, length of the dorsolateral spines; DMSL, length of the most dorsal spines), we used the terminology presented by Maldonado et al. (2001a). Data obtained were compared with descriptions available to Echinostoma spp. reported in South America (Lie and Basch, 1967;Travassos et al., 1969;Maldonado et al., 2001aMaldonado et al., , 2003Fernandes et al., 2015).
The type material is deposited in the Helminthological Collection of the Institute Oswaldo Cruz (CHIOC) and Collection of Trematodes of the Federal University of Minas Gerais (UFMG-TRE).
were carried out in a final volume of 25 μL, including 12.5 μ,L of Platinum Hot Start Master Mix 2 × (Invitrogen, Thermo Fisher Scientific Baltics, Vilnius, Lithuania), 1.25 pmol of each primer, about 50 ng of template DNA and sterile ddH 2 O. PCR products were electrophoresed on a 1.5% agarose gel, and the band of the expected size was purified with 20% polyethylene glycol 8000 (Promega, Madison, WI, USA). Purified DNA was sequenced in both directions by capillary electrophoresis in an ABI3730 sequencer using the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA). The chromatograms obtained were assembled using ChromasPro v.2.0.1 software (Technelysium Pty Ltd, South Brisbane, Queensland, Australia) and the generated contiguous sequences used in the subsequent analyses.
The newly generated sequences were first subjected to a search in the Basic Local Alignment Search Tool -BLAST (Altschul et al., 1990) (https://blast.ncbi.nlm.nih.gov) in GenBank. Data obtained for the 4 molecular markers evaluated were compared exclusively with congeneric species. Alignment constructions were performed using the algorithm MUSCLE implemented in the software MEGA X (Kumar et al., 2018). Before phylogenetic analyses, alignments were trimmed to match the shortest sequence. The evolutionary models used for the analyses were estimated by the Bayesian information criterion implemented in MEGA X. Outgroups for each dataset were chosen based on previous molecular phylogenetic studies including representatives of Echinostoma and related taxa (Sereno-Uribe et al., 2015;Tkach et al., 2016;Georgieva et al., 2017;Nakao and Sasaki, 2021). The methods used to generate the phylogenetic hypotheses were maximum likelihood (ML) and Bayesian inference (IB). ML analyses were performed in MEGA X and nodal support was measured using the bootstrap test with 1000 replicates. BI analyses were performed with MrBayes v.3.2.6 software (Ronquist et al., 2012) and posterior probability values were determined by the Monte Carlo method via Markov chains, in 2 simultaneous runs of 4 chains for 10 6 generations, with topologies sampling every 100 generations. The 'burn-in' was established for the first 25% of the trees sampled, and the nodal support was estimated from the remaining trees. The trees obtained by BI were visualized with FigTree software version 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/) and edited in Adobe Photoshop® v.22.4.3 software (Adobe Inc., USA). All sequences generated in this study were deposited in GenBank under the accession numbers OQ132569 (28S), OQ132538 (ITS1-5.8S-ITS2), OQ134393 (cox1) and OQ126877 (nad1).

Results
A total of 56 specimens of a 37-collar-spined Echinostoma were recovered from the small intestine of M. unguiculatus 15 days after the experimental infection with metacercariae, obtained from S. marmorata. The obtained morphological and molecular data pointed to a new species of Echinostoma from the 'revolutum' group, which is described below.

Description
Adult (n = 24, Figs 1 and 2; measurements in Table 1): Body slender, elongate, dorsoventrally flattened, with maximum width at uterine level, 4.5-6.4 times longer than wide, with almost parallel lateral margins and notable constriction at posterior margin of ventral sucker. Tegument with scale-like spines in alternating transverse rows, denser at pharynx region, extending to level of posterior margin of ventral sucker and reaching posterior testis level on ventral surface. Forebody short, 9-15% of body length. Head collar (n = 10) well developed, reniform, width representing 18-34% of maximum body width. Collar spines conspicuous, 37 in number in most specimens (5 with 37, 2 with 33, 1 with 36, 38 or 39 spines), with following arrangement: 5 angle spines (3 oral and 2 aboral); 6 lateral spines in single row on each ventral lappet, and 15 dorsal spines in double row (8 oral and 7 aboral). Dorsal spines varying in length, with the dorsolateral spines markedly larger than central (dorsalmost) spines. Oral sucker ventrosubterminal, rounded, muscular. Prepharynx very short; pharynx oval to elongate-oval, muscular. Oesophagus short, 2-4% of body length; oesophageal bifurcation just anterior to ventral sucker; intestinal caeca blind, almost reaching posterior extremity. Ventral sucker spherical, muscular, located in the first quarter of body; 2 times as large as oral sucker (suckers-width ratio 1:2.1-2.4). Testes 2, subglobular, smooth or slightly indented, in tandem, located in third quarter of body; post-testicular region (between posterior testis and posterior extremity) corresponding to 25-44% of body length. Cirrus sac elongate-oval, anterior to ventral sucker, with a simple elongate-oval seminal vesicle, welldeveloped pars prostatica, coiled ejaculatory duct and unspined cirrus. Genital pore at the level of anterior margin of ventral sucker. Ovary transversely oval, post-equatorial, pretesticular. Mehlis' gland contiguous with ovary. Uterus intercaecal, with numerous coils between ventral sucker and ovary; uterine field corresponding to 24-32% of body length. Metraterm short, weakly muscular. Vitellarium follicular, forming 2 lateral nonconfluent fields of small follicles overlapping caeca and extending from level of the region slightly posterior to ventral sucker to posterior extremity of body. Eggs abundant, oval, yellow, operculated, immature.

Larval stages
Mature redia (n = 23, Fig. 3A; Table 2): Body elongate, tapered at its anterior and posterior ends, with yellowish pigment. Collar absent. Pharynx subspherical, muscular. Caecum short, with dark pigment inside. Birth pore prominent at the anterior region, lateral. Two prominent locomotor appendages present at about 2/ 3 of body length.
Penetration glands with granular content, preacetabular, medially distributed, along oesophagus, with canaliculi directed to anterior region, opening in 6 ducts at extremity of oral sucker (Fig. 3D). Paraesophagean glands absent. Cystogeneous glands with     small, at posterior extremity of body. Main ducts of excretory system presenting numerous, small calcareous concretions (about 50, 6-9 μm of diameter) between the pharynx and ventral sucker. Details of excretory system, including flame-cell formula, position of excretory pore and caudal excretory duct not characterized. Metacercaria (n = 35, Fig. 3E; Table 2): cysts spherical, on average 135 ± 7 μm in diameter; cyst walls composed of 2 layers, the outer layer transparent, about 6 μm thick, and the inner layer opaque, thin, about 2 μm thick.

Remarks
The experimentally recovered adult specimens can be readily distinguished from all but 1 species of Echinostoma of the 'revolutum' complex by bearing tiny dorsal collar spines, much smaller than the dorsolateral and lateral ones (see description). Despite the differences in the head collar commented above, the recently described E. pseudorobustum Valadão et al., 2022 presents similar size and body shape. However, this species is differentiated by having a longer forebody (FORE% c. 18.9 vs 12), oesophagus length as a proportion of body length (OSL% c. 10 vs 3), head collar as a proportion of body width (CW/BW c. 49.5 vs 26.1%) and uterine field (OVAR c. 41.6 vs 28%).
Taking into account the morphological data alone, we initially assigned the new taxon to E. paraensei because this species is the only one in the 'revolutum' complex that also possesses tiny dorsal collar spines (Kostadinova and Gibson, 2000), and also due to similar morphometrics comparing other key morphological features (see Table 1). The geographical origin of the specimens studied (Mesoregion of Belo Horizonte) also led us to preliminarily identify the isolate as E. paraensei. There are some differences observed between the newly collected specimens and those previously identified as E. paraensei by other workers, e.g. body shape (BW%,, ratio between the width of oral and ventral suckers (OSW:VSW, 1:2.5 vs 1:1.4-2.8) and prepharynx length (PL) (PL: 41 vs 72-120 μm in the original description). These differences are attributed here to the evaluation of specimens with different ages (range of 12-109 dpi) in each study as individuals continue to grow after reaching maturity and the use of different techniques for fixing the material (flattening or not of the specimens).
While morphological features suggested the conspecificity of E. maldonadoi n. sp. and E. paraensei, molecular data, specifically based on the mitochondrial nad1 gene, revealed interspecific levels of nucleotide divergence between the 2 species (see below).

Molecular study
Partial sequences of the 28S rRNA gene (1197 bp), ITS1-5.8S-ITS-2 region (1159 bp), cox1 gene (415 bp) and nad1 gene (478 bp) were successfully generated for the isolate of 37-collar-spined E. maldonadoi n. sp. Phylogenetic trees were reconstructed using BI and ML criteria, but only those obtained through the first approach are shown, including values of nodal support for both analyses. The trimmed 28S alignment was 1051 bp long and included 18 sequences for 14 species of the 'revolutum' group of Echinostoma. The phylogenetic reconstruction based on this gene (Fig. 4A) revealed that E. maldonadoi n. sp. clustered in a well-supported clade with an isolate of E. paraensei maintained under laboratory conditions in the USA, without nucleotide differences between the sequences. The interspecific variation in relation to other Echinostoma spp. included in the analyses was 0.3-1.1%.
A dataset containing 9 species of Echinostoma was included in the analysis of the nuclear region ITS1-5.8S-ITS2 (trimmed alignment: 1001 bp). Echinostoma maldonadoi n. sp. grouped with 3 Brazilian isolates of E. paraensei in a strongly supported clade (97/0.98) (Fig. 4B), but the actual phylogenetic affinities with these isolates remained unclear. Moreover, the molecular divergence in relation to the 4 isolates of E. paraensei from N. squamipes was low (0-0.1%). Representatives of this clade appeared as sister to isolates of E. trivolvis, but the support was low. The interspecific genetic variation in relation to other Echinostoma spp. included in the analyses was 0.7-2.6%.
The molecular data obtained for the faster evolving mitochondrial genes (nad1 and cox1) revealed nucleotide differences between the new species and E. paraensei. For cox1 gene, a trimmed alignment (366 bp) containing 8 species of Echinostoma was analysed. The resolution of the tree was poor resulting in a polytomy, which hampered our understanding on the phylogenetic relationships of most taxa (Fig. 5A). Nevertheless, E. maldonadoi n. sp. was recovered in a strongly supported clade together with E. paraensei. Moreover, in the cox1 tree, E. maldonadoi n. sp. + E. paraensei is a sister group to E. caproni, and this nesting was well-supported by BI (0.96). This clade forms a polytomy with E. cinetorchis, E. miyagawai and 2 'E. revolutum' isolates. The genetic divergence between E. maldonadoi n. sp. and the laboratory isolate of E. paraensei from the USA was 3.8% (difference at 14 nucleotide positions), which might suggest that they do not belong to the same species. The molecular divergence in relation to other Echinostoma spp. ranged from 10.9 to 15.9%.
The molecular analysis based on the nad1 gene unequivocally confirmed the distinct species status of E. maldonadoi n. sp. The trimmed alignment (438 bp) included 153 sequences of 19 species/species-level lineages of Echinostoma. In the well-resolved phylogenetic tree (Fig. 5B), the new species grouped in a strongly supported clade with 3 isolates of E. paraensei, and both appeared as sister clade to E. nasincovae (albeit with no support in the ML analysis). The genetic divergence in relation to the isolate of E. paraensei maintained under laboratory conditions in the USA, and in relation to another isolate of the same species from Brazil, reported by Morgan and Blair (1998b), was 6.2% (for 2 isolates) and 6.4% (27 and 28 nucleotides), respectively, which fell into the interspecific range of variability for Echinostoma spp. of the 'revolutum' group. Moreover, the newly described species exhibited a genetic divergence of 12.3-21.0% compared with the other species of the 'revolutum' group. Echinostoma maldonadoi n. sp. can be diagnosed based on unique sites/nucleotides in specific positions of the nad1 alignment comparing to isolates of E.

Discussion
Our understanding of the species diversity of the genus Echinostoma has been improved by integrating molecular and morphological approaches to better delimit species boundaries and consequently address more accurately questions concerning host use, geographical distribution and life cycle (Georgieva et al., 2013(Georgieva et al., , 2014Faltýnková et al., 2015;Izrailskaia et al., 2021;Valadão et al., 2022). A number of studies stressed the presence of several cryptic lineages within the 'revolutum' species complex of Echinostoma, mostly from the Holarctic realm (Detwiler et al., 2010(Detwiler et al., , 2012Georgieva et al., 2014;Izrailskaia et al., 2021), which presented challenges either for identification of known taxa or the proposal of new taxa with no morphological differential trait to report. Even though cryptic diversity is more common among trematodes than other groups of parasitic helminths (Pérez-Ponce de León and Poulin, 2018), some of the cryptic evolutionary independent lineages found in previous studies of Echinostoma spp. may be found morphologically identifiable, once all developmental stages (primarily adults) are described. In the present study, the integration of morphological, molecular and life cycle data made it possible to propose a new species of Echinostoma, E. maldonadoi n. sp., cryptically related to E. paraensei considering all developmental stages, yet some morphometric differences were found between isolates of the new species and E. paraensei (see remarks).
From the biological point of view, the intermediate host use is not considered a significant difference between E. paraensei and E. maldonadoi n. sp. Although natural infection for the former was reported in planorbid snails, B. grabrata and Glyptophysa sp., in Brazil and Australia, respectively (Lie and Basch, 1967;Morgan and Blair, 1998a), the experimental susceptibility of physid snails to E. paraensei was reported to the isolate from Belo Horizonte (original description) (Lie and Basch, 1967) and for the isolate from Sumidouro, state of Rio de Janeiro, Brazil (Maldonado et al., 2001b). Nevertheless, echinostome cercariae were not found among 767 specimens of Biomphalaria spp. (including B. glabrata) collected in Alvorada de Minas (Coelho et al., 2021), the type locality of E. maldonadoi n. sp., which may suggest that E. paraensei and E. maldonadoi n. sp. are primarily found in planorbid and physid snails, respectively, at least in natural conditions.
A question that emerges from this study, but that is impossible to be answered at the current stage of the knowledge, is whether the subsequent reports of E. paraensei-like specimens really correspond to this taxon or E. maldonadoi n. sp., or even to another new cryptic species of the genus. Interestingly, some relevant differences between isolates identified as E. paraensei were reported regarding: the time of miracidial development, worm burden, number of uterine eggs and parasite distribution in the site of infection (Maldonado et al., 2001a(Maldonado et al., , 2001b. The possibility that such differences may represent interspecific variation instead of just evident phenotypic plasticity, as previously thought , cannot be ruled out. In this sense, future nad1 sequencing of this laboratory-maintained isolate of E. paraensei is here encouraged for taxonomic validation of the parasite species used in the different experimental studies previously published (Ferraz et al., 2012;Gonçalves et al., 2013;Monte et al., 2016Monte et al., , 2018Monte et al., , 2019Souza et al., 2017b).
The utility of the most densely sampled nuclear ribosomal genetic regions, 28S and ITS, and mitochondrial protein-coding genes, cox1 and nad1, were evaluated in molecular phylogenetic studies Blair, 1998a, 1998b;Georgieva et al., 2014;Izrailskaia et al., 2021). While all of them agree that mitochondrial genes should be the first choice for barcoding species of Echinostoma in the 'revolutum' group, as nuclear ribosomal genes may not provide enough phylogenetic signal to differentiate closely related species, they differ concerning which mitochondrial marker is more informative. Most of the scientific contributions provided compelling evidence that nad1 shows a sufficient gap between levels of intraspecific and interspecific genetic divergence, the so-called barcode gap, allowing the identification of species boundaries (Morgan and Blair, 1998b;Georgieva et al., 2014Georgieva et al., , 2017. For this reason, most taxonomic surveys on Echinostoma provided nad1 sequences (Georgieva et al., 2014(Georgieva et al., , 2017Faltýnková et al., 2015;Pantoja et al., 2021). However, Izrailskaia et al. (2021) argued that cox1 may be more informative than nad1 and might solve more confidently taxonomic problems within the Echinostomatidae.
In the present study, we reinforce the limitations of ribosomal nuclear DNA for the distinction of closely related species (the new taxon could not be recognized using this dataset) and found only moderate nucleotide divergence (3.83%) using cox1 between the sister taxa E. maldonadoi n. sp. and E. paraensei, much lower than the 6.3-13.7% and 7.3-16.8% interspecific values reported by Morgan and Blair (1998b) and Izrailskaia et al. (2021), respectively. These different values may just be biased by the paucity of cox1 sequences for Echinostoma spp., leaving just a few sequences of closely related sister taxa to be compared. Contrary to that, the divergence values (6.2-6.4%) of the nad1 marker between the new species and E. paraensei were within the interspecific range of variability for Echinostoma spp. of the 'revolutum' group. For instance, in the most comprehensive study involving molecular taxonomy and species delineation in Echinostoma spp., Georgieva et al. (2014) recognized 17 species/species-level lineages using nad1 dataset and found 4.2-21.5% of interspecific divergence. Moreover, identification was achieved for all monophyletic lineages at 3% divergence threshold. These same authors found a mean intraspecific divergence of 0.2-1.8%. More recently, Pantoja et al. (2021) reported 0-1.6% of intraspecific divergence for Echinostoma revolutum s.str. from Europe and 0.5-1.2% for isolates identified as Echinostoma sp. IG. Thus, the divergence found between E. maldonadoi n. sp. and E. paraensei is more than double from the higher intraspecific divergence previously reported for species of Echinostoma. It is also worth noting, that the nucleotide divergence using nad1 between E. maldonadoi n. sp. and the remaining species of the 'revolutum' group (12.3-21.0%) was considerably higher than compared with its sister taxon (E. paraensei), which may indicate a very recent speciation event and segregation into these 2 morphologically indistinguishable species.
Our findings support the conclusions of Morgan and Blair (1998b) and Georgieva et al. (2014) that the best mitochondrial marker for the investigation of phylogenetic interrelationships and identification of cryptic diversity within the 'revolutum' group of Echinostoma is nad1. In fact, most internal or terminal clades are strongly supported in our phylogenetic reconstruction. Moreover, the availability of nad1 datasets with larger taxonomic and geographical coverage in relation to the remaining markers allows the comparison of genetic distances between 2 putative species with values available for 'benchmark' congeners. Nevertheless, finding a 'genetic yardstick' remains challenging in trematodes (Nadler and Pérez-Ponce de León, 2011) and even in Echinostoma.
Even though the reciprocal monophyly of E. maldonadoi n. sp. and E. paraensei could not be assessed as only a single haplotype of the new taxon was herein detected, the knowledge leveraged for the molecular taxonomy of this genus in the last 2 decades, including a clear barcode gap using nad1 dataset, suffices for our new species proposal. This hypothesis though may be strengthened by molecular characterization of new isolates and widening the number of target molecular markers, such as encompassing the entire mitochondrial genome.