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GENETIC DISTANCE AND PHYLOGENETIC RELATIONSHIPS OF SOME ECHINOSTOMA SPECIES ( E. MALAYANUM, E. REVOLUTUM , E. MIYAGAWAI ) AND HYPODERAEUM CONOIDEUM (FAMILY ECHINOSTOMATIDAE) INFERRED FROM PARTIAL 28S rDNA SEQUENCE ANALYSIS

Echinostomiasis is a neglected disease caused by the intestinal flukes (family Echinostomatidae, suborder Echinostomata) and is common in communities in Asian countries, such as India, Indonesia, the Philippines, China, Malaysia, Singapore, Korea, Japan, Thailand, Laos, Cambodia and Vietnam. The genetic markers from the nuclear ribosomal transcription units are commonly used in genetic studies and phylogenetic analyses. A portion of the 28S rDNA sequence (domains D1–D3, of 1062–1067 bp for the final use) was obtained from the zoonotic Echinostoma malayanum (strain E/Amala-EMI3-TH), E. revolutum (strain Erevo-MSD15-TH), E. miyagawai (Emiya-RED11-TH), and Hypoderaeum conoideum (Hcono-RED42-TH) species; and used to perform an alignment for genetic distance estimation and phylogenetic analysis. The alignment was performed using 62 strains of 42 species from 19 genera of the family Echinostomatidae, including Echinoparyphium , Echinostoma , Artyfechinostomum , Patagifer, Neoacanthoparyphium, Hypoderaeum, Echinoparyphium, Drepanocephalus, Euparyphium, Chaunocephalus , Neopetasiger, Ribeiroia, Cathaemasia , Rhopalias , Isthmiophora , Petasiger, Moliniella, Pegosomum , and Schistosoma (family Schistosomatidae). The genetic distance estimation among 16 strains/10 species has shown a low intra-specific divergence level between strains within the same species, such as E. miyagawai (0–0.10%), E. revolutum (0.10–0.50%), and H. conoideum (0–0.10%), while between strains within the same genus it was higher (normally over 1.0%) and among strains/species between genera it was the highest (3.06–4.12%). The 28S rDNA sequence analysis and phylogenetic relationship well supported the Echinostoma/ Artyfechinostomum malayanum intergeneric taxonomy and the topology indicated clear, well-supported positions of member species in different genera in the family Echinostomatidae of the suborder Echinostomata. More sensu lato samples of the genera, are required for sequencing, particularly those of zoonotic species in the five genera: Artyfechinostomum , Echinostoma , Hypoderaeum , Echinoparyphium , and Isthmiophora . The resultant mitochondrial and nuclear data obtained from these species will be a good source to use to clearly assess the taxonomic and generic relationships.

The major challenge in taxonomy of echinosomes and Echinostomatidae systematics is a gap needing to be filled in the understanding of evolutionary and phylogenetic relationships of species within the family Echinostomatidae of the suborder Echinostomata (Tantrawatpan et al., 2013;Georgieva et al., 2013;2014;Faltýnková et al., 2015;Chai, 2009;2019). DNA sequences are commonly used for molecular diagnosis, providing a basis for the development of accurate diagnostic tools and systematic/phylogenetic studies. The use of molecular markers has solved the specific classification and phylogenetic relationships of particular species of Echinostoma and genera within the family Echinostomatidae, and between families of the suborder Echinostomata (Kostadinova, 2005;Georgieva et al., 2014;Tkach et al., 2016;Chai, 2019;Chai, Jung, 2020).
The DNA markers from the nuclear ribosomal transcription unit (rTU) (including 18S, ITS1, ITS2, and 28S) have been shown to be crucial in resolving taxonomic issues for parasitic worms (Tkach et al., 2016;Le et al., 2020). The 18S and 28S rDNA sequences as well as the intergenic regions (ITS-1, ITS-2) were used as reliable molecular markers in the analysis of phylogenetic and molecular evolutionary relationships between species and taxonomic classification (Weider et al., 2005;Blair, 2006;Tkach et al., 2016;Pérez-Ponce de León et al., 2019). A detailed ribosomal phylogenetic analysis of taxonomically confused echinostomes, particularly those related to Echinostoma/Artyfechinostomum and Hypoderaeum and their generic congeners, will facilitate clarification of inter-relationships among species of the family Echinostomatidae (and Echinostomata suborder).
Thus, the aim of this paper is to present the use of 28S rDNA sequences to assess the genetic distance and an in-depth phylogenetic approach to resolve the interrelationship between E. malayanum (synonym: Artyfechinostomum malayanum), E. miyagawai, E. revolutum, H. conoideum and their trematode congeners in the genera Echinostoma, Artyfechinostomum, and Hypoderaeum in the Echinostomatidae of the suborder Echinostomata. This study involved four strains of four Echinostomatidae species, E. malayanum (E/Amala-EMI3-TH), E. revolutum (Erevo-MSD15-TH), E. miyagawai , and H. conoideum (Hcono-RED42-TH) ( Table 1). Due to its synonymy with the generic name of Artyfechinostomum (as it is Artyfechinostomum malayanum) (Chai, 2019;Pham et al., 2022), the abbreviation of the E. malayanum strain, is changed to E/Amala-EMI3-TH for use.

MATERIALS AND METHODS
Total genomic DNA was extracted from an individual worm, using the DNA extraction kit (QIAGEN, Hilden, Germany) as instructed, eluted in 100 μL, and stored at −20°C until use (Saijuntha et al., 2011;Tantrawatpan et al., 2013). The DNA concentration was estimated using a Thermo Scientific Nanodrop 1000 UV/VIS spectrophotometer and diluted to a working 50 ng/μL and 2 μL was used as a template in a PCR of 50 μL volume.

Primers, amplification and sequence annotation
Primers, including forward LSU1F (5' CTTAAGCATATCACTAAGCGGAGG 3') and reverse LSU3R (5′ GCTATCCTGAGGGAAACTTCG 3′) were designed based on the alignment of 28S rDNA sequences available from GenBank and previous publications. These primers were used for amplification of a 28S rDNA region (i.e., the D1-D3 domain) to obtain a PCR amplicon of about 1.3 kb in length, and they were also used as the flanking primers for direct sequencing from both ends. PCR reactions of 50 μL were prepared using 25 μL of DreamTaq PCR Master Mix (2X) (Thermo Fisher Scientific Inc., MA, USA) and 2 μL of DNA template (50 ng/μL), 2 μL of each primer (10 pmol/μL), 2 μL DMSO (dimethyl sulphoxide) and 17 μL of water, performed in an MJ PTC-100 thermal cycler. Initiation was at 94°C for 5 min, followed by 35 cycles consisting of denaturation for 30 s at 94°C, annealing at 52°C for 30 s, extension at 72°C for 6 min, and a final extension at 72°C for 10 min. The PCR products (10 μL of each) were examined on a 1% agarose gel, stained with ethidium bromide and visualized under UV light (Wealtec, Sparks, NV, USA). The amplicons were sent to the sequencing services (Nam Khoa company, Ho Chi Minh City) for direct sequencing.
The partial 28S rDNA sequence for each of the four Echinostomatidae species was obtained after editing chromatograms (using Chromas 2.6.6; http://technelysium.com.au/wp/chromas/) and was 1120-1230 bp for analysis, respectively, and deposited in the GenBank database (NCBI).

Sequence analysis
A total of 62 partial 28S rDNA sequences, approximately 1.  Table 1). The final alignment block containing sequences of 1062-1067 bp was used for estimation of genetic distance and assessment of phylogenetic relationships. Table 1. List and information on strains and species that provide partial nuclear ribosomal 28S rDNA sequences for phylogenetic analysis and tree construction for the assessment of the taxonomic relationships of the family Echinostomatidae (Trematoda: Platyhelminthes).

No
Species Abbrevi -ation Note: Species: full name; abbreviation: five letters with the first capital letter from the genus and next four are the first letters from the species name. Sequence designation: the strain name is in between the abbreviation of the species and the country names (two letters) in which the bolded names indicate the sequences obtained in this study. *Outgroup sequence (from Schistosoma haematobium (Schistosomatidae)).

Genetic distance estimation
A pairwise distance analysis was also performed and estimated as a measure of genetic distance (p-distance) between 16 strains of 10 species of three genera (Echinostoma, Artyfechinostomum, and Hypoderaeum) in the family Echinostomatidae. The 16 sequence alignment was imported into MEGA X. The analysis was set for distance estimation using the "Maximum Composite Likelihood" model/method with 1000 bootstrap replications (Kumar et al., 2018).

Phylogenetic reconstruction
To examine the phylogenetic position of the Echinostomatidae species relative to their congeners and other trematodes, a phylogenetic tree was reconstructed from the alignment of 62 partial 28S rDNA sequences (listed in Table 1), including the outgroup sequence, using the maximum likelihood (ML) analysis in the MEGA X program. The substitution model with the best score according to the Bayesian information criterion was the (GTR+G+I) model, with residue frequencies estimated from the data (GTR), rate variation along the length of the alignment (+G), and allowing for a proportion of invariant sites (+I) (Kumar et al., 2018).
Overall, a relatively high divergence was seen between E. revolutum (Erevo-MSD15-TH and Erevo-VVT2015-US) at 3.80%, while the highest divergence was between A. malayanum and H. conoideum , at 4.12%. Between E/Amala-EMI3-TH of Echinostoma/ Artyfechinostomum malayanum and Asufr-Shillong-IN (KF781303) of Artyfechinostomum sufrartyfex, the genetic distance was shown at 1.0%, which was too low in respect of an interspecific (between species) variation level. It was debated that the extremely close interrelationship between E. malayanum (or A. malayanum) and A. sufartyfex makes it possible to consider as an intraspecific variation level. Or indeed, as to which taxonomic validity is for this fluke, Echinostoma malayanum, or Artyfechinostomum sufartyfex, or Artyfechinostomum malayanum or all should be unified into one. The generic name for this species has been recently suggested to be retaken by Artyfechinostomum malayanum based on the complete mitochondrial genome analysis (Pham et al., 2022).

Phylogenetic interrelationships and taxonomic position of Echinostoma, Artyfechinostomum, and Hypoderaeum species
To examine the phylogenetic interrelationships and taxonomic position of some Echinostoma, Artyfechinostomum, and Hypoderaeum species in the family Echinostomatidae within the suborder Echinostomata, an ML tree was constructed from a phylogenetic analysis of 62 partial 28S rDNA sequences for 43 trematode species belonging to 18 genera of the family Echinostomatidae and an outgroup species, Schistosoma haematobium (Schistosomatidae) (Table 1; Fig. 1).
In the tree presented in Fig. 1, 62 sequences were placed in 21 groups/branches that were distinguished from each other. Besides the outgroup sequence (S. haematobium), the majority of groups/branches were clearly performed from the sequences of strains/species of 18 genera they belong to, including Echinoparyphium, Echinostoma, Artyfechinostomum, Patagifer, Neoacanthoparyphium, Hypoderaeum, Echinoparyphium, Drepanocephalus, Euparyphium, Chaunocephalus, Neopetasiger, Ribeiroia, Cathaemasia, Rhopalias, Isthmiophora, Petasiger, Moliniella, and Pegosomum. The topology indicated well in the phylogenetic tree ( Fig. 1), that the genus Echinostoma with E. miyagawai (strain Emiya-RED11-TH) and E. revolutum (strain Erevo-MSD15-TH) in this study was placed as a "sister" group to Artyfechinostomum, formed by E/Amala-EMI3-TH of Echinostoma/ Artyfechinostomum malayanum and Asufr-Shillong-IN of Artyfechinostomum sufrartyfex. The E. malayanum species was resolved as a sister taxon to A. sufrartyfex and was confirmed as a member of the genus Artyfechinostomum. The group of Hypoderaeum conoideum, including the strain Hcono-RED42-TH of this study, was rendered as paraphyletic (Fig. 1). Table 2. Pairwise genetic distances (%) among 16 strains/10 species and between the genera of Echinostoma, Artyfechinostomum, and Hypoderaeum estimated based on the analysis of the partial 28S rDNA sequences.  Note: Information for strain/ species is given in Table 1. The sequences for the strains/species of this study are bolded (Nos 11,14,22,32). The different highlighted blocks in the first column (E/Amala-EMI3-TH) and the last row (Hcono-NA-US) indicate the pairwise genetic divergence between these species and members of each genus. The intra-specific genetic distance of strains within each species (E. miyagawai, E. revolutum, H. conoideum) is squared. Figure 1. A maximum likelihood phylogenetic tree showing the interrelationships and taxonomic position of Echinostoma, Artyfechinostomum, and Hypoderaeum in this study (solid circle symbol) and other members of the family Echinostomatidae based on analysis of the partial 28S rDNA sequence data from 62 strains/species. This tree was reconstructed by the MEGA X program (Kumar et al., 2018)   However, there are some exceptions. The positions of the Echinoparyphium aconiatum (Eacon-(Lstag)-CZ) and Euparyphium melis (Emeli-UA(sub)) species and the Petasiger and Pegosomum genera were the exceptions. According to the topology, Echinoparyphium aconiatum was removed from the cluster of Echinoparyphium species (being placed as paraphyletic from Echinoparyphium and the Hypoderaeum group was bracketed in), and Euparyphium melis was removed from the Euparyphium cluster (was placed in the Isthmiophora group). Another interesting placement is that the genus Pegosomum was bracketed between two clades of the genus Petasiger clusters (Table 1; Fig. 1). The mispositions may be involved in the taxonomic misidentification of these species and the wrong deposition in GenBank.

DISCUSSION
The Echinostomatidae are one of the largest families in the class Trematoda that exhibits an extremely high level of diversity. The morphological characters that have been used for species identification and taxonomic analysis are not sufficient to clarify the closely related taxa (Georgieva et al., 2013;2014;Faltýnková et al., 2015). This has been a particular issue for the genus Echinostoma since there are multiple synonyms and newly described species, leading to frequent revision of Echinostomatidae systematics (Tantrawatpan et al., 2013;Chai, 2009;2019). The spine collar examination can often be a tenuous characteristic for species differentiation, and the exact criteria for species taxonomy and family systematics have not been determined (Georgieva et al., 2014;Tkach et al., 2016;Chai 2019;Le et al., 2020).
The phylogenetic analyses have resolved well-supported monophyletic clusters for the majority of genera, especially for the genera Echinostoma, Artyfechinostomum, and Hypoderaeum. The validity of the generic Artyfechinostomum for Echinostoma malayanum has been solved to retake the generic name of Artyfechinostomum malayanum, as previously suggested (Mehlhorn, 2015;Sohn et al., 2017;Chai, 2019), and recently confirmed by mitochondrial sequence analysis (Pham et al., 2022). The 28S rDNA sequence analysis indicates that the family Echinostomatidae presents broad systematic and taxonomic challenges and validates the combination of using morphological and molecular, both mitochondrial and ribosomal genomic datasets to ensure the taxonomic and generic relationships of member species in the family Echinostomatidae of the suborder Echinostomata (Olson et al., 2003;Tkach et al., 2016;Chai, 2019;Pérez-Ponce de León et al., 2019;Le et al., 2020;Pham et al., 2022). To clarify the genetic and taxonomic situations, more sensu lato samples of the genera are required for sequencing, particularly those of zoonotic species in the five genera: Artyfechinostomum, Echinostoma, Hypoderaeum, Echinoparyphium, and Isthmiophora. The resultant mitochondrial and nuclear data obtained from these species will be a good source to use to clearly assess the taxonomic and generic relationships.

CONCLUSION
In conclusion, the present study determined the genetic distance and phylogenetic interrelationships of Echinostoma/Artyfechinostomum malayanum (E/Amala-EMI3-TH), Echinostoma miyagawai (Emiya-RED11-TH), Echinostoma revolutum (Erevo-MSD15-TH), and Hypoderaeum conoideum species with other echinosome species from 21 genera in the family Echinostomatidae. The genetic distance estimation has shown a low intraspecific rate between strains within the same species, E. miyagawai, E. revolutum and H. conoideum. The 28S rDNA sequence analysis and phylogenetic relationship well supported the Echinostoma/Artyfechinostomum malayanum intergeneric taxonomy and the clear, wellsupported positions of member species in the family Echinostomatidae in the suborder Echinostomata.