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Two trematode species, Pseudoparabascus khotenovskii comb. n. and Acanthatrium ovatum (Trematoda: Microphalloidea), from East Asia: morphological and molecular data

Published online by Cambridge University Press:  04 May 2026

Y.V. Tatonova Open the ORCID record for Y.V. Tatonova [Opens in a new window]  and
V.V. Besprozvannykh
Y.V. Tatonova*
Affiliation:
Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch, Russian Academy of Sciences , pr-t 100-letiya Vladivostoka 159a, Vladivostok 690022, Russia
V.V. Besprozvannykh
Affiliation:
Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch, Russian Academy of Sciences , pr-t 100-letiya Vladivostoka 159a, Vladivostok 690022, Russia
*
Corresponding author: Y.V. Tatonova; Email: ytatonova@gmail.com
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Abstract

In this report, morphological and molecular characteristics are provided for two members of the subfamily Microphalloidea distributed in the south of the Russian Far East. Some of trematode specimens found in larvae of mayflies, stoneflies, and caddisflies and then experimentally reared to the adult stage in hamsters, according to their morphological features, belong to the species Acanthatrium ovatum previously recorded from Japanese bats. Molecular data has confirmed their membership in the family Lecithodendriidae and similarity to trematodes identified as Lecithodendriidae sp. F from Japan. The other specimens, experimentally reared in chickens, according to their morphological characteristics, meet the diagnostic criteria of the genus Parabascus. However, this species is not clustered with representatives of this taxon in the phylogenetic reconstruction based on 28S rRNA gene data. A molecular analysis has shown that it forms a single cluster with Pachypsolus irroratus and is a member of the family Pachypsolidae. It is here placed in the new genus Pseudoparabascus n. g. under the name Pseudoparabascus khotenovskii comb. n. The phylogenetic reconstructions based on two other markers, the cox1 mtDNA gene and the ITS2 rDNA region, have confirmed the assignment of these specimens obtained in both experiments to the above-considered taxonomic groups.

Keywords

PseudoparabascusAcanthatriumRussiamorphologymolecular data

Information

Type
Research Paper
Information
Journal of Helminthology , Volume 100 , 2026 , e44
Copyright
© The Author(s), 2026. Published by Cambridge University Press

Introduction

Trematodes of the speciose superfamily Microphalloidea Ward, 1901, parasitize mammals, birds, reptiles, and amphibians. Members of this taxonomic group have a number of morphological features that are shared by not only different species but also different genera within this superfamily. For this reason, the superfamily Microphalloidea has been repeatedly revised, first by Morozov (Reference Morozov1955) and, for the last time, by Bray (Reference Bray, Bray, Gibson and Jones2008). In most cases, the authors’ conclusions were based on morphological characteristics of mature flukes. Tkach and co-authors (Reference Tkach, Littlewood, Olson, Kinsella and Świderski2003) carried out one of the most significant revisions of Microphalloidea using a data set including both morphological characteristics of trematodes from this superfamily, information about their definitive hosts, and molecular markers. Subsequently, the superfamily has been extended by adding a significant number of new taxa on the basis of both morphological and molecular data (Kanarek et al. Reference Kanarek, Zalesny, Sitko and Tkach2014, Reference Kanarek, Zaleśny, Sitko and Tkach2017; Bell et al. Reference Bell, González-Acuña and Tkach2018; Pérez-Ponce de León and Hernández-Mena Reference Pérez-Ponce de León and Hernández-Mena2019; Sokolov et al. Reference Sokolov, Shchenkov, Kalmykov and Smirnova2020; Fernandes et al. Reference Fernandes, Dos Santos, de Vasconcelos Melo, Achatz, McAllister, Bonilla and Tkach2022; Kirillova et al. Reference Kirillova, Shchenkov, Kirillov and Ruchin2022; Vlasenkov et al. Reference Vlasenkov, Kalmykov and Sokolov2023, etc.). However, although numerous taxa represent the superfamily in East Asia, the vast majority of molecular data are available for microphalloideans found outside the region (Besprozvannykh et al. Reference Besprozvannykh, Ermoolenko and Nadtochiy2012; Ito Reference Ito, Morishita, Komiya and Matsubayashi1964; Le et al. Reference Le, Ngo and Ermolenko2013; Yamaguti Reference Yamaguti1971). In order to fill this gap, as well as to expand the knowledge about taxa that were not taken into account in phylogenetic reconstructions due to the lack of molecular data, we conducted experimental studies. We managed to obtain morphological and molecular characteristics for mature flukes of two species from Microphalloidea inhabiting the south of the Russian Far East. The results of the study are presented below.

Materials and methods

Parasitological material and morphological data

To rare mature individuals of Microphalloidea, we used larvae of mayflies (Heptageniidae Needham, 1901), stoneflies (Perlidae Latreille, 1802), and caddisflies (Semblis spp.) naturally infected by trematodes of this superfamily. The insect larvae were collected with a scoop net in the Komissarovka River, Primorsky Krai, Russian Far East. In this locality, we previously found snails Parajuga spp. that emitted cercariae morphologically identical to Parabascus khotenovskii Besprozvannykh, Reference Besprozvannykh2000, and Acanthatrium ovatum Yamaguti, 1993 (Besprozvannykh Reference Besprozvannykh1990, Reference Besprozvannykh2000). Dissection of the insects revealed 2 to 15 metacercariae per larva similar to the above-mentioned species in the adult stage. Ten infected mayflies, 10 stoneflies, and 5 caddisflies were fed to each of 2 hamsters (Mesocricetus auratus) and 2 chickens (Gallus gallus dom.) kept in the laboratory. At 8 days post-infection (dpi), we found 7 and 12 adult specimens in the hamsters’ intestines; at 10 dpi, 4 and 10 adult trematode specimens in the chickens’ intestines. We examined the specimens under a microscope using the temporal slide preparation technique, rinsed them in saline, and fixed in 70% ethanol. After fixation, they were placed in 96% ethanol. Whole mounts for morphological descriptions of the specimens were made by staining them with alum carmine, dehydrating in a graded ethanol series, clearing in clove oil, and mounting in Canada balsam on a glass slide under a coverslip. All measurements are in micrometres (μm).

The parasitological material obtained in this study was deposited in the Bioresource Collection of the Federal Scientific Center of the East Asia Terrestrial Biodiversity (FSCEATB), Far East Branch, Russian Academy of Sciences (FEB RAS) (reg. number 2797657, hereinafter referred to as ‘Bioresource Collection’); email: .

We also used specimens of trematodes identified as Parabascus khotenovskii Besprozvannykh, Reference Besprozvannykh2000 (Holotype No. 1-Tr and paratypes No. 2-3-Tr) and Acanthatrium ovatum Yamaguti, Reference Yamaguti1939 (No. 1248) and deposited in the Bioresource Collection.

Molecular data acquisition and analysis

We used one trematode specimen from an infected hamster and one from a chicken for genetic analysis. We extracted genomic DNA from the adult specimens using the HotSHOT technique (Truett et al. Reference Truett, Heege, Mynatt, Truett, Walker and Warman2000). Partial nucleotide sequences of the 28S rRNA gene and ITS2 rDNA region were amplified and sequenced using the following primers: forward digl2 (5′-AAG-CAT-ATC-ACT-ACT-CGG-3′) and reverse 1500R (5′-GCT-ATC-CTG-AGG-GAA-ACT-TCG-3′) (Tkach et al. Reference Tkach, Littlewood, Olson, Kinsella and Świderski2003) for the 28S rRNA gene, and BD1 (5′-GTC-GTA-ACA-AGG-TTT-CCG-TA-3′) (Luton et al. Reference Luton, Walker and Blair1992) and 28S4R (5′-TAT-TTA-GCC-TTG-GAT-GGA-GTT-TAC-C-3′) (Besprozvannykh et al. Reference Besprozvannykh, Tatonova and Shumenko2019) for the ITS region. Additionally, the ribosomal gene was sequenced using internal primers 900F (5′-CCG-TCT-TGA-AAC-ACG-GAC-CAA-G-3′) and 1200R (5′-CTT-GGT-CCG-TGT-TTC-AAG-ACG-GG-3′) (Tkach et al. Reference Tkach, Littlewood, Olson, Kinsella and Świderski2003). Partial sequences of the cox1 gene were amplified and sequenced using primers JB3 (5′-TTT-TTT-GGG-CAT-CCT-GAG-GTT-TAT-3′) and JB4.5 (5′-TAA-AGA-AAG-AAC-ATA-ATG-AAA-ATG-3′) (Bowles and McManus Reference Bowles and McManus1994). The reaction mixture (with a total volume of 10 μL) contained 5 μL GoTaq® Green Master Mix, 0.5 μL of each of the primers, and 3 μL DNA. The reaction mixture for sequencing (10 μL) contained 1.0 μL of BigDye Terminator v3.1, 0.5 μL of primers, and 1.0 μL of PCR product. Determination of primary nucleotide sequences was carried out on an Applied Biosystems 3500 Genetic Analyzer at the Instrumental Center for Biotechnology and Gene Engineering, FSCEATB FEB RAS (Vladivostok, Russia).

We visually checked the sequences obtained using the Finch TV ver. 1.4.0 program and aligned them in the MEGA ver. 11.0.13 program (Tamura et al. Reference Tamura, Stecher and Kumar2021). In the study, we also used sequences of other members of the superfamily Microphalloidea accessed from the NCBI database (Table 1). We selected three specimens of the trematode family Plagiorchioidea (AF151932, Lecithopyge rastellus; MH041375, Paramacroderoides echinus; and JX522535, Plagiorchis elegans) as an outgroup. Short sequences were excluded from the analysis. The length of the aligned 28S rRNA gene sequences with indels was 1,123 bp. For the phylogenetic analysis based on the other two sequences (the cox1 gene and ITS2 rDNA region), we relied on data obtained using the 28S rRNA marker and used only the sequences from the species and families that appeared in the same clusters with the species considered in this study (representatives of the families Pachypsolidae and Lecithodendriidae), as well as with the species from the cluster that included representatives of the genus Parabascus. Representatives of the genus Renicola were used as outgroups (Tables 2, 3). The lengths of the aligned sequences of the cox1 gene and ITS2 region with indels were 287 and 216 bp, respectively. The p-distances between and within species were analyzed in the MEGA software without including indels.

Table 1.

List of Microphalloidea species used in the analysis of 28S rRNA gene sequences and other associated information

Note: The new sequences obtained in this study are highlighted in bold.

Table 2.

List of Microphalloidea species used in the analysis of the ITS2 rDNA region sequences and other associated information

Note: The new sequences obtained in this study are highlighted in bold.

Table 3.

List of Microphalloidea species used in the analysis of the cox1 mtDNA gene sequences and other associated information

Note: The new sequences obtained in this study are highlighted in bold.

We reconstructed phylogenetic relationships by the Bayesian inference (BI) algorithm in MrBayes 3.1.2 (Ronquist and Huelsenbeck Reference Ronquist and Huelsenbeck2003) using the TVM+I+G models that were selected based on Akaike’s informational criterion (AIC) as optimal for the 28S rRNA gene in the jModeltest 2.1.7 program (Darriba et al. Reference Darriba, Taboada, Doallo and Posada2012). The TPM2uf+G model was optimal for the other two markers. We estimated statistical significance of phylogenetic relationships for the trees constructed using the BI algorithm by the a posteriori probability method. In the Bayesian analysis, 10,000,000, 700,000, and 2,400,000 generations of Markov chain Monte Carlo (MCMC) were implemented for the 28S rRNA gene, the cox1 gene, and the ITS2 region, respectively. The number of generations was considered sufficient, because the SD value calculated was ˂0.01. The chain was sampled every 100 generations. Of the samples assessed, 25% were excluded to permit construction of consensus trees.

Results and discussion

We found that the adult trematode specimens experimentally reared in hamsters, according to their morphological features, belonged to the species Acanthatrium ovatum previously recorded from Japanese bats. The molecular data confirmed their membership in the family Lecithodendriidae and similarity to trematodes identified as Lecithodendriidae sp. F from Japan. The other specimens experimentally reared in chickens, according to their morphological characteristics, met the diagnostic criteria of the genus Parabascus. However, they were not clustered with representatives of this taxon in the phylogenetic reconstruction based on 28S rRNA gene data. A molecular analysis showed that they formed a single cluster with Pachypsolus irroratus and were members of the family Pachypsolidae. We placed them in the new genus Pseudoparabascus n. g. under the name Pseudoparabascus khotenovskii comb. n. The phylogenetic reconstructions based on two other markers, the cox1 mtDNA gene and the ITS2 rDNA region, confirmed the assignment of these specimens to the two above-mentioned taxonomic groups. Below are morphological descriptions, ecological, molecular data, and results of phylogenetic analyses for the trematode specimens under study.

Pachypsolidae (Yamaguti, 1958)

Pseudoparabascus khotenovskii comb. n.

(Syn. Parabascus khotenovskii Besprozvannykh, Reference Besprozvannykh2000)

Taxonomic summary

Host. Gallus gallus dom. (infected experimentally).

Site. Intestine.

Intensity of infection; 4 and 10 specimens per individual.

First intermediate hosts. Snails Parajuga spp. (Semisulcospiridae, Gastropoda).

Second intermediate hosts. Mayflies Heptageniidae, stoneflies Perlidae, and caddisflies Semblis spp.

Locality. Komissarovka River, Primorsky Krai, southern Far East of Russia (44°49′N, 131°33′E).

This material (adult trematodes, whole mounts, Nos. 1249–1253) was deposited in the Bioresource Collection on 5 May 2025; email: .

Molecular data. One partial sequence of the 28S rRNA gene (GenBank accession No. PX387198); one partial sequence of the ITS2 region of nuclear DNA (No. PX925803); and one partial sequence of the cox1 gene of mitochondrial DNA (No. PX780645).

Description

Adult flukes (based on five specimens; Table 4, Figure 1a). Body inverted, pear-shaped. Anterior half of body wide, posterior half narrowed. Tegument with spines from anterior end to level of testes. Oral sucker subterminal, rounded or slightly transversely oval. Pharynx transversely oval, adjacent to oral sucker. Oesophagus short. Intestinal bifurcation located anteriorly to ventral sucker. Caeca almost reaching posterior end of body. Ventral sucker rounded, smaller than oral sucker, located in middle third of body. Testes two, symmetrical, elongate-oval, located in middle third of body. Anterior end of testes at level of ventral sucker. Cirrus-sac curved at level of intestinal bifurcation. Distal end of cirrus-sac covered by ventral sucker. Internal seminal vesicle elongate-saccular. Prostatic cells few. Genital pore submedian, at level of ventral sucker and sinistrally to it. Ovary rounded, located dextrally to ventral sucker, at level of intestinal bifurcation and ventral sucker. Ovary partly covered by anterior end of dextral testis. Seminal receptacle between testes, at level of posterior end of ventral sucker. Uterus post-testicular, with loops extending between testes. Eggs numerous. Vitellarium with two lateral fields extending from level of posterior end of oral sucker to anterior ends of testes. Follicles rounded or irregular in shape. Excretory vesicle V-shaped. Excretory pore terminal.

Table 4.

Morphometric parameters of adult Microphalloidea trematodes (μm)

Note: dpi, days post-infection

Figure 1.

Adult flukes (hand drawings): (a) Pseudoparabascus khotenovskii comb. n.; (b) Acanthatrium ovatum.

Remark

The mature specimens we obtained had morphological characteristics that matched the diagnostic criteria of Parabascus Looss, 1907 (Lotz and Font Reference Lotz, Font, Bray, Gibson and Jones2008). Among members of this genus, they were most similar in all morphological characteristics to the specimens designated by Besprozvannykh (Reference Besprozvannykh2000) as Parabascus khotenovskii. Some differences in size (Table 2) may be due to the different times of residence of both in the definitive hosts. Previous studies (Besprozvannykh Reference Besprozvannykh2000) showed that the trematodes designated as P. khotenovskii had sporocyst stages in snails Parajuga spp. from which virgulate xiphidiocercariae emerged. These cercariae infect larvae of mayflies and caddisflies in which metacercariae are formed. The mature stage of trematode undergoes development in the bird intestine (Besprozvannykh Reference Besprozvannykh2000). Among other representatives of the genus Parabascus, the trematode specimens in our material were most similar to Parabascus indicus (Agrawall, 1968) in their morphological features. The only difference between them, except metric parameters, was the position of the testes relative to the ventral sucker: the anterior end of the testes at the level of the ventral sucker in the specimens we obtained vs. the testes posteriorly to the ventral sucker in P. indicus (Agrawall, 1968). Moreover, one of the most important indicators of their different taxonomic groups is the involvement of endothermic vs. ectothermic animals as definitive hosts, respectively. Considering the morphology of all developmental stages, the trematodes in our study matched the descriptions of members of two families, Phaneropsolidae Mehra, 1935, and Pleurogenidae Looss, 1899 (Lotz and Font Reference Lotz, Font, Bray, Gibson and Jones2008). However, according to Lotz and Font (Reference Lotz, Font, Bray, Gibson and Jones2008), the sets of definitive hosts differ between these families: in the Phaneropsolidae, these are mammals and birds that are preferred less frequently than reptiles, while in the Pleurogenidae, amphibians are more frequently selected as definitive hosts than reptiles. Trematodes P. khotenovskii parasitize birds and, therefore, they presumably belong to the family Phaneropsolidae to which Lotz and Font (Reference Lotz, Font, Bray, Gibson and Jones2008) assigned members of the genus Parabascus. However, shortly before this conclusion, an analysis based on molecular data showed that Parabascus species belong to the family Pleurogenidae (Tkach et al. Reference Tkach, Lotz, Świderski and Esteban2002, Reference Tkach, Littlewood, Olson, Kinsella and Świderski2003) which was considered as a separate group of bat parasites. Subsequently, the placement of this genus in the Pleurogenidae was confirmed by Kanarek et al. (Reference Kanarek, Zalesny, Sitko and Tkach2014), Bell et al. (Reference Bell, González-Acuña and Tkach2018), and Sokolov et al. (Reference Sokolov, Shchenkov, Kalmykov and Smirnova2020). As already mentioned above, the trematodes we obtained were identical in their morphology to the trematodes identified as Parabascus khotenovskii and were, apparently, part of Pleurogenidae.

However, the use of the 28S rRNA gene showed that the trematodes we obtained got in a single cluster with Pachypsolus irroratus within the family Pachypsolidae (Yamaguti, 1958) in the phylogenetic reconstruction (Figure 2). However, all Parabascus species in the phylogenetic tree formed a separate, well-differentiated branch within the family Pleurogenidae and were not related to the Far Eastern parasite. The p-distance between Pachypsolus irroratus and the trematodes in our material was 9.3%, which was equivalent to the intergeneric level of differences between members of the superfamily (Supplementary Material 1). These trematodes belonged to different genera, and the morphological characteristics such as the location and structure of the vitellarium confirmed their differences: the vitellarium with follicles located on the sides of the body from the pharynx to the posterior half of the body (Pachypsolus irroratus) vs. the vitellarium with follicle clusters in the anterior half of the body (trematodes obtained in this study). The trematode group, combined with the specimens from our material, included specimen LC599542 whose data was obtained at the sporocyst stage from the snail Semisulcospira libertina in Japan. There were also two specimens (MH094413 and MH094432) at larval stages from the snail Pleurocera proxima in the USA. The genetic distances between them were in a range of 1.3–1.9%, which suggested interspecific differences within a genus. One of the characteristic features of this trematode group was the involvement of snails from the family Cerithioidea Fleming, 1822 as the first intermediate hosts in their life cycles. This fact may also indicate a close relationship between these trematodes. There is also data for Cercaria cattieni emitted from cerithioids in Vietnam, which were placed in Pachypsolidae (Krupenko et al., Reference Krupenko, Gonchar, Kremnev, Efeykin and Krapivin2020). However, in the tree, this trematode occupied a position far from the pachypsolids and differed from the latter by 13–15% (p-distances). Based on these data, it can be assumed with confidence that Cercaria cattieni does not belong to the family Pachypsolidae. Thus, the results of our experimental study indicate that the trematodes we obtained are morphologically identical to the specimens previously designated as Parabascus khotenovskii by Besprozvannykh (Reference Besprozvannykh2000). They do not belong to Parabascus despite their morphological similarity with other members of the genus. Based on their differences at the molecular level from other representatives of Microphalloidea, we here place the trematode known as Parabascus khotenovskii in a new, separate genus Pseudoparabascus n. g. within the Pachypsolidae. The genus should also include specimens LC599542, MH094413, and MH094432.

Figure 2.

Phylogenetic relationships within the superfamily Microphalloidea inferred from analysis of partial 28S rRNA gene sequences (1,123 bp) by the Bayesian inference (BI) method under the TVM+I+G model. Posterior probabilities more than 0.50 are indicated for the respective clades. Coloured boxes indicate two families, Pachypsolidae (green) and Lecithodendriidae (yellow). The new sequences obtained in this study are highlighted in bold.

The short sequence length of the ITS2 rDNA region hinders identification of the relative positions of most groups and branches using this marker in a phylogenetic reconstruction (Figure 3). However, Pseudoparabascus khotenovskii formed a highly supported group with two unidentified trematodes (LC805637 and LC805638), differing from them by 1.2% (Supplementary Material 2). Therefore, this marker cannot confirm the membership of the studied species in the family Pachypsoliidae. However, Parabascus duboisi (MN726994), which clustered with other species of the genus Parabascus in the 28S rRNA gene tree (Figure 2), occurred in a different clade, thus relating two other representatives of the family Pleurogenidae. Pseudoparabascus khotenovskii differed from all these species by 8.9–14%. It is premature to judge whether these genetic differences for the ITS2 marker are sufficient to discriminate between the families Pleurogenidae and Pachypsolidae due to the lack of data for representatives of both taxa. However, these values are sufficient to state that P. khotenovskii and P. duboisi are representatives of different genera.

Figure 3.

Phylogenetic relationships within the superfamily Microphalloidea inferred from analysis of partial ITS2 rDNA region sequences (216 bp) by the Bayesian inference (BI) method under the TPM2uf+G model. Posterior probabilities more than 0.50 are indicated for the respective clades. Coloured boxes indicate two families, Pachypsolidae (green) and Lecithodendriidae (yellow). The new sequences obtained in this study are highlighted in bold.

Unfortunately, data on the mitochondrial cox1 gene is scarce and is completely unavailable for the genus Parabascus (Figure 4). However, P. khotenovskii again clustered with a specimen from Japan (LC599899). In the 28S reconstruction, this specimen (corresponding to LC599542) also clustered with P. khotenovskii (Figure 2). In the tree inferred from the analysis of this mitochondrial marker, the sequence OR230530 of Lecithodollfusia arenula occupied an external position relative to both specimens which we previously assigned to family Pachypsoliidae based on the nuclear marker. The genetic p-distances between the two pachypsoliids were multifold lower than the differences between them and L. arenula (the family Pleurogenidae according to Vlasenkov et al., Reference Vlasenkov, Kalmykov and Sokolov2023, Supplementary Material 3). This suggests that all three markers work in concert, but there is a lack of actual data for analysis.

Figure 4.

Phylogenetic relationships within the superfamily Microphalloidea inferred from analysis of partial cox1 mtDNA gene sequences (287 bp) by the Bayesian inference (BI) method under the TPM2uf+G model. Posterior probabilities more than 0.50 are indicated for the respective clades. Coloured boxes indicate two families, Pachypsolidae (green) and Lecithodendriidae (yellow). The new sequences obtained in this study are highlighted in bold.

Pseudoparabascus n. g

Body inverted pear-shaped. Anterior half of body wide, posterior half narrowed. Tegument with spines. Oral sucker subterminal. Ventral sucker in middle third of body. Pharynx transversely oval. Oesophagus short. Intestinal bifurcation located anteriorly to ventral sucker. Caeca almost reaching posterior end of body. Testes symmetrical, in middle third of body. Cirrus-sac curved at level of intestinal bifurcation. Distal end of cirrus-sac covered by ventral sucker. Internal seminal vesicle elongate-saccular. Prostatic cells few. Genital pore submedian, located at level of ventral sucker and sinistrally to it. Ovary rounded, located dextrally to ventral sucker, at level of intestinal bifurcation and ventral sucker. Seminal receptacle at level of posterior end of ventral sucker. Uterus post-testicular, with loops extending between testes. Eggs numerous. Vitellarium extending from level of posterior end of oral sucker to anterior end of testes. Excretory vesicle V-shaped. Excretory pore terminal. In intestine of birds. First intermediate hosts: snails Cerithioidea. Type species: Pseudoparabascus khotenovskii.

Molecular characteristics of Pseudoparabascus n. g. for differentiating the species from the genus Parabascus, expressed in terms of genus-specific substitutions at different nucleotide positions in alignments for 28S rRNA gene sequences, are presented in the following format: Ns, where N is the nucleotide and s is the site of this nucleotide (A is adenine; T, thymine; G, guanine; and C, cytosine; Supplementary Material 4).

  1. (i) Molecular characteristics of Pseudoparabascus n. g. (based on Pseudoparabascus khotenovskii PX387198; we did not use sequences LC599542, MH094413, and MH094432 here, since there was a complete lack of description of morphological criteria for these individuals, and they were not identified even to the family level)

    T32, T71, T113, G117, C119, A128, G138, A171, C278, A331, C374, A377, C411, G437, T470, T479, A480, C486, C524, G552, T567, G605, T610, T619, A623, G629, T632, T637, C639, A716, A728, T730, C736, A742, T746, G771, G774, G781, C788, C816, A819, A898, T959, A962, T1049, T1050.

  2. (ii) Molecular characteristics of Parabascus (based on Parabascus semisquamosus ON005555–ON005557, AF151923; Parabascus joannae AY220619; Parabascus lepidotus ON036091–ON036094; and Parabascus duboisi AY220618, MN726967)

    C32, C71, A113, T117, T119, C128, A138, G171, T278, G331, T374, G377, T411, A437, A470, G479, G480, T486, T524, T552, C567, A605, C610, C619, G623, A629, G632, A637, T639, T716, G728, C730, T736, G742, C746, A771, A774, A781, T788, T816, T819, G898, C959, G962, C1049, C1050.

Lecithodendriidae Lühe, 1901

Acanthatrium ovatum Yamaguti, Reference Yamaguti1939

Taxonomic summary

Host. Mesocricetus auratus (infected experimentally).

Site. Intestine.

Intensity of infection; 7 and 12 specimens per individual.

First intermediate hosts. Snails Parajuga spp. (Semisulcospiridae, Gastropoda).

Second intermediate hosts. Mayflies Heptageniidae, stoneflies Perlidae, and caddisflies Semblis spp.

Locality. Komissarovka River, Primorsky Region, the Russian southern Far East; 44°49′N, 131°33′E.

This material (adult flukes, whole mounts, Nos. 1254–1258) was deposited in the Bioresource Collection on 5 May 2025; email: .

Molecular data. One partial sequence of the 28S rRNA gene (GenBank accession No. PX387199; one partial sequence of the ITS2 region of nuclear DNA No. PX925804; and one partial sequence of the cox1 gene of mitochondrial DNA (No. PX780646).

Description

Adult flukes (based on five specimens, Table 4, Figure 1b). Body wide, oval, with narrowed anterior and posterior ends. Tegument armed with spines from anterior end to level of testes. Oral sucker subterminal, rounded. Pharynx transversely oval, adjacent to oral sucker. Oesophagus short. Intestinal bifurcation at middle of forebody. Caeca short, reaching anterior ends or middle of testes. Ventral sucker rounded, equatorial, equal to or smaller than oral sucker. Testes two, symmetrical, rounded or elongate-oval, at level of ventral sucker and pseudocirrus sac. Pseudocirrus sac rounded or oval, largely between intestinal bifurcation and ventral sucker or largely covered by ventral sucker. Internal seminal vesicle filling most of pseudocirrus sac. Prostatic cells few. Genital atrium armed with two groups of spines: needle-shaped spines and wedge-shaped spines. Genital pore median, located immediately posteriorly to intestinal bifurcation. Ovary rounded, located dextrally to ventral sucker and partially covering posterior edge of dextral testis. Ovary partly covered by anterior end of dextral testis. Mehlis glands located immediately sinistrally to ovary. Uterus post-testicular. Eggs numerous. Vitellarium forming two lateral fields extending from level of posterior end of oral sucker to anterior ends of testes. Follicles oval or irregular in shape. Excretory vesicle V-shaped. Excretory pore terminal.

Remark

In morphometric characteristics (Table 2), the specimens from our material were identical to Acanthatrium ovatum from Japanese bats (Yamaguti Reference Yamaguti1939) and were obtained as a result of studying the life cycle of trematodes found in snails Parajga spp. from the Russian southern Far East (Besprozvannykh Reference Besprozvannykh1990). In our study, we obtained molecular data from species of the genus Acanthatrium for the first time. The sequence of A. ovatum in the phylogenetic reconstruction based on 28S rRNA was found in a single group with nine other specimens, most of which had undetermined generic memberships within Lecithdendriidae (Figure 3). The exception was a specimen of Paralecithodendrium parvouterus (Bhalerao, 1926) that had an external position relative to the rest of this group and differed from them by 3.0–4.5%, including a 4% difference from A. ovatum in the present study. Other sequences within the group differed from A. ovatum by 0.3–3.2%. The closest to A. ovatum was sequence LC704422 (Lecithodendriidae sp. F) obtained for the sporocyst stage found in Semisulcospira libertina in Japan (unpublished data). It was intriguing that the snails of the superfamily Cerithioidea served as the first intermediate hosts both for specimen LC704422 and for A. ovatum. In the trees based on the two other markers, A. ovatum also clustered with Japanese specimens designated as Lecithodendriidae sp. F (Figures 3, 4), with, however, no differences from them in the ITS2 rDNA region (Supplementary Material 2). Differences in the mitochondrial cox1 gene were also low, amounting to only 1.4% (Supplementary Material 3), which suggested a close relationship between these trematodes. Nevertheless, the available morphological and molecular data do not provide unambiguous answers as to the taxonomic identity of the sporocyst stage from Japan and the degree of its relationship with A. ovatum. This uncertainty stems from the fact that, although Japan and the continental part of the Russian southern Far East are inhabited by morphologically similar trematodes, which can be assigned to A. ovatum, molecular data for mature specimens from Japan are not available. The presence of even low differences at the molecular level from the specimens found in the larval stage suggests that these may be different species, as the genetic distances between them matched differences at the species level. Moreover, we previously found cryptic species from other taxonomic groups of trematodes from Japan and the Russian Far East that either differed slightly in the phylogenetic tree based on the 28S rRNA gene or had no differences (Shumenko et al. Reference Shumenko, Tatonova and Besprozvannykh2017; Solodovnik et al. Reference Solodovnik, Tatonova, Urabe, Besprozvannykh, Nakao and Inoue2021; Tatonova et al. Reference Tatonova, Shumenko and Besprozvannykh2018). Thus, based on the morphological similarity with Acanthatrium ovatum, we assign the trematode specimens we found to this species and provide a detailed morphological description and molecular data that can subsequently clarify the status of Japanese samples.

Conclusion

The above-described challenges of taxonomic identification of only two species show that, despite large sets of molecular data regularly obtained for the superfamily Microphalloidea, many issues concerning the multilevel taxonomy and phylogenetic relationships remain unresolved. However, the large amounts of molecular data for trematodes within the Lecithodendriidae and other families obtained from larval stages can be of little use without morphological characteristics of adults. The lack of such information complicates genus and species identification. Moreover, most of molecular data are available as sequences in the databases and not supported by publications with species descriptions. Thus, it sometimes becomes almost impossible to determine which values of genetic distances correspond to interspecific, intergeneric, or higher taxonomic levels. For this reason, based on the available morphological and molecular data for Microphalloidea, we have provided new data only for two species without a broad discussion of phylogenetic relationships within the superfamily, which we consider premature.

Supplementary material

The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X26101461.

Acknowledgements

The experiments described in this study were set up using the equipment at the Instrumental Center for Biotechnology and Gene Engineering, Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch, Russian Academy of Sciences (Vladivostok, Russia).

Financial support

The research was carried out within the framework of the State assignment from the Ministry of Science and Higher Education of the Russian Federation (topic No. 124012400285-7).

Competing interests

The authors declare no conflict of interest.

Ethical standard

Animals (hamsters and chickens) were euthanized in accordance with the resolution of the Committee on the Ethics of Animal Experiments, Federal Scientific Center of the East Asia Terrestrial Biodiversity, FEB RAS, Russia (Permit no. 1 of 25 April 2022). 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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Figure 0

Table 1. List of Microphalloidea species used in the analysis of 28S rRNA gene sequences and other associated information

Figure 1

Table 2. List of Microphalloidea species used in the analysis of the ITS2 rDNA region sequences and other associated information

Figure 2

Table 3. List of Microphalloidea species used in the analysis of the cox1 mtDNA gene sequences and other associated information

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Table 4. Morphometric parameters of adult Microphalloidea trematodes (μm)

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Figure 1. Adult flukes (hand drawings): (a) Pseudoparabascus khotenovskii comb. n.; (b) Acanthatrium ovatum.

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Figure 2. Phylogenetic relationships within the superfamily Microphalloidea inferred from analysis of partial 28S rRNA gene sequences (1,123 bp) by the Bayesian inference (BI) method under the TVM+I+G model. Posterior probabilities more than 0.50 are indicated for the respective clades. Coloured boxes indicate two families, Pachypsolidae (green) and Lecithodendriidae (yellow). The new sequences obtained in this study are highlighted in bold.

Figure 6

Figure 3. Phylogenetic relationships within the superfamily Microphalloidea inferred from analysis of partial ITS2 rDNA region sequences (216 bp) by the Bayesian inference (BI) method under the TPM2uf+G model. Posterior probabilities more than 0.50 are indicated for the respective clades. Coloured boxes indicate two families, Pachypsolidae (green) and Lecithodendriidae (yellow). The new sequences obtained in this study are highlighted in bold.

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Figure 4. Phylogenetic relationships within the superfamily Microphalloidea inferred from analysis of partial cox1 mtDNA gene sequences (287 bp) by the Bayesian inference (BI) method under the TPM2uf+G model. Posterior probabilities more than 0.50 are indicated for the respective clades. Coloured boxes indicate two families, Pachypsolidae (green) and Lecithodendriidae (yellow). The new sequences obtained in this study are highlighted in bold.

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