Morphological and molecular characterization of plagiorchiid trematodes (Plagiorchis: Plagiorchiidae, Digenea) from bats with redescription of Plagiorchis mordovii Shaldybin, 1958

Abstract

Five Plagiorchis spp. parasitize bats in European Russia: Plagiorchis elegans, Plagiorchis koreanus, Plagiorchis mordovii, Plagiorchis muelleri, and Plagiorchis vespertilionis. Their identification is difficult due to a high morphological similarity. The morphological variability of these species is poorly studied. The taxonomic position of P. mordovii remains debatable. The purpose of our study was to analyse Plagiorchis spp. from European bats using a combination of morphological and molecular-phylogenetic approaches and to establish the taxonomic position of the problematic species P. mordovii. Plagiorchis spp. were shown to be variable morphologically and morphometrically both from various host species and from different specimens of the same species. We presented a new taxonomic key for identification of the Plagiorchis spp. from European bats, provided a complete description of Plagiorchis mordovii, and confirmed the validity and the generic affiliation of this species.

Introduction

The family Plagiorchiidae Lühe, 1901 is one of the largest in the superfamily Plagiorchidea. The most common mammals that serve as definitive hosts of its type genus Plagiorchis Lühe, 1899 are bats (Chiroptera). Five Plagiorchis spp. are known from bats: Plagiorchis elegans (Rudolphi, 1802); Plagiorchis koreanus Ogata, 1938; Plagiorchis mordovii Shaldybin, 1958; Plagiorchis muelleri Tkach & Sharpilo, 1990; and Plagiorchis vespertilionis (Müller, 1784) (Sharpilo and Iskova 1989; Tkach and Sharpilo 1990; Sharpilo and Tkach 1992; Tkach et al. 2000b; Kirillov et al. 2012a, 2012b, 2015, 2017; Ruchin et al. 2016).

Plagiorchis vespertilionis was described by Müller (1780, 1784) as Fasciola vespertilionis from the brown long-eared bat, Plecotus auritus (Linnaeus, 1758), in Denmark. However, this author provided only a brief description and a schematic drawing, so this species cannot be reliably distinguished from other Plagiorchis spp. According to Tkach and Sharpilo (1990) and Sharpilo and Tkach (1992), almost all Plagiorchis spp. found in bats have been identified as P. vespertilionis. This was partly due to confusion in several reviews, where other species were described as P. vespertilionis or where the species description was based on mixed material (Braun 1900; Dawes 1946; Odening 1964; Skrjabin and Antipin 1958; Yamaguti 1958, 1971; Groschaft and Tenora 1973, 1974; Krasnolobova 1987). Tkach et al. (2000b) differentiated Plagiorchis spp. from bats using molecular data and provided a redescription of P. vespertilionis.

Plagiorchis vespertilionis is a common parasite of bats found in many European countries (Odening 1964; Zdzitowiecki 1970; Matskasi 1967, 1973; Skvortsov 1972; Chiriac and Barbu 1973; Bakke and Mehl 1977; Tkach and Sharpilo 1990; Sharpilo and Tkach 1992; Ricci 1995; Sumer and Yildirimhan 2017, 2018, 2019; Shimalov 2021). In Russia, this species is known from bats in the Astrakhan region (Kurochkin and Kurochkina 1962), the Samara region (Demidova and Vekhnik 2004; Kirillov et al. 2012a, 2012b, 2017), Mordovia (Schaldybin 1964; Kirillov et al. 2015; Ruchin et al. 2016; Kirillova et al. 2018, 2023), Karelia (Lebedeva et al. 2020), and Kamchatka (Orlovskaya and Dokuchaev 2017).

Shaldybin in Skrjabin and Antipin (1958) described Plagiorchis mordovii from Myotis dasycneme (Boie, 1825) and Plagiorchis symmetrica Schaldybin, 1958 from Vespertilio murinus Linnaeus, 1758. Krasnolobova (1977) considered these digenean species to be identical and synonymized P. symmetrica with P. mordovii. In addition, she transferred this species to the new genus Symmetricatesticula Krasnolobova, 1977. Tkach (2008) doubted the validity of Symmetricatesticula because, in his opinion, the descriptions of the species within it were incomplete. According to Tkach (2008), Plagiorchis mordovii (P. symmetrica in Tkach) is closer to the pleurogenids than to the plagiorchiids. After the original description, Plagiorchis mordovii was recorded only in M. dasycneme in Poland, in Myotis brandtii (Eversmann, 1845), M. dasycneme, V. murinus, and Nyctalus noctula (Schreber, 1774) in Russia (Shaldybin 1964; Zdzitowiecki 1970; Demidova and Vekhnik 2004; Kirillov et al. 2012a, 2012b, 2015, 2017; Ruchin et al. 2016; Kirillova and Kirillov 2017; Kirillova et al. 2018, 2023).

Plagiorchis muelleri was described from different bats in Ukraine (Tkach and Sharpilo 1990). Tkach and Sharpilo (1990), on the basis of their own material and literature data, found that one more Plagiorchis sp. parasitizes bats of Europe in addition to the known ones. This species has a wide Palearctic distribution. According to Tkach and Sharpilo (1990), earlier researchers apparently identified it as P. vespertilionis. In our opinion, it is more likely that this species was identified as P. koreanus (Braun 1900; Rysavy 1956; Kochseder 1969; Zdzitowiecki 1970; Skvortsov 1972; Groschaft and Tenora 1973; Meszaros and Mas-Coma 1980; Alvarez et al. 1991; Shimalov et al. 2011; Naumkin and Sivkova 2019). Plagiorchis muelleri is known from Eptesicus serotinus Schreber, 1774 in Belarus (Shimalov et al. 2002; Shimalov 2021), from Myotis aurescens Kuzyakin, 1935 and Myotis daubentonii (Kuhl, 1817) in Turkey (Sumer and Yildirimhan 2017, 2018, 2019), from M. brandtii in the Samara region (Demidova and Vekhnik 2004), and from P. auritus and M. brandtii in Karelia (Lebedeva et al. 2020). We found P. muelleri in bats of the Samara region and Mordovia (Kirillova et al. 2007, 2023; Kirillov et al. 2012a, 2012b; 2015; Ruchin et al. 2016). Plagiorchis muelleri has not been found in other countries.

Plagiorchis koreanus, broadly distributed in the Palearctic, has the widest range of bat hosts (Sharpilo and Iskova 1989; Kirillov et al. 2012a, 2012b; Ruchin et al. 2016). This species was found (as P. vespertilionis) in bats from Georgia (Matsaberidze and Khotenovsky 1967), in Rhinolophus ferrumequinum (Schreber, 1774) (as Plagiorchis sp.) from Spain (Esteban et al. 1999), in bats from Italy (Lanza 1999), in Myotis spp. from Turkey (Sumer and Yildirimhan 2017, 2018, 2019), and in the United Kingdom from Pipistrellus spp. (Lord et al. 2012). In Russia, P. koreanus was recorded in various bats in the Samara region (Demidova and Vekhnik 2004; Kirillov et al. 2012a, 2012b; Kirillova and Kirillov 2017), Mordovia (Kirillov et al. 2015; Ruchin et al. 2016; Kirillova et al. 2018, 2023), Karelia (Lebedeva et al. 2020) and in P. auritus and Myotis sibiricus Kastschenko, 1905 from the Magadan region (Gulyaev et al. 2002; Orlovskaya et al. 2022).

Plagiorchis elegans is found in a wide range of vertebrate hosts (birds, mammals, and reptiles) but is especially common in passerine birds (Sharpilo and Iskova 1989; Kirillov et al. 2012a). Plagiorchis elegans is the least common species of this genus in bats. It was found in Myotis alcathoe (von Helversen and Heller 2001) and M. daubentonii from Turkey (Sumer and Yildirimhan 2019). We found P. elegans in P. auritus from the Samara region and in V. murinus and Nyctalus noctula (Schreber, 1774) from Mordovia (Kirillova et al. 2007, 2023; Kirillov et al. 2012a, 2012b; Kirillova and Kirillov 2017).

In this work, we studied four Plagiorchis spp. (P. koreanus, P. mordovii, P. muelleri, and P. vespertilionis) from bats in the Middle Volga region (European Russia). The aim of our study was to provide a reliable identification of Plagiorchis spp. parasitizing bats by combining morphological and morphometric analyses with the newly obtained molecular phylogenetic data and to validate the species Plagiorchis mordovii.

Materials and methods

Trematode collection and examination

Adult Plagiorchis specimens were collected from dead bats in two localities in the European Russia: near Shiryaevo village (Samarskaya Luka National Park, Samara region) in 2005 and 2006 and near Smolny village (Smolny National Park, Republic of Mordovia) in 2019. In total, we examined 14 bat specimens that belonged to four species: five M. brandtii, four M. daubentonii, two N. noctula, and three Eptesicus nilssoni Keyserling & Blasius, 1839. No animals were killed intentionally for our research. Several dead bats were kindly provided by the staff of the Smolny National Park. Several bats that died of natural causes during wintering or were killed by cats were provided by local residents.

Only alive motile mature trematodes were collected for further study. For the morphological examination, the trematodes were recovered from the bat intestine and killed by heating in water. Then they were stained with aceto-carmine, dehydrated in an ethanol series (70–96%), cleared in clove oil, and mounted in Canada balsam (Ivashkin et al. 1971; Anikanova et al. 2007). Plagiorchis specimens for molecular phylogenetic analysis were fixed in 96% ethanol and stored at +4°C for further evaluations. In total, 90 trematode specimens were studied: Plagiorchis koreanus (30), Plagiorchis muelleri (15), Plagiorchis vespertilionis (15), and Plagiorchis mordovii (30). Data on the geographic origin of the examined Plagiorchis trematodes are presented in Table 1.

Table 1. List of Plagiorchis specimens, with GenBank accession numbers according to the geographic origin

Drawings of digeneans were made using the Levenhuk M500 BASE Digital Camera and drawing tube RA-7 attached to MBI-9 light microscope. All measurements are given in millimeters. Literature information for the comparative analysis was taken only from the published works that contained drawings and morphometric data on the Plagiorchis spp. under study. The trematodes were identified according to Tkach et al. (2000b), Skrjabin and Antipin (1958), Zdzietowiecki (1970), Sharpilo and Iskova (1989), and Kirillov et al. (2012a). Voucher specimens are stored in the parasitological collection of the Institute of Ecology of the Volga River Basin of the Russian Academy of Sciences (Togliatti, Russia).

DNA extraction, amplification, sequencing, and phylogenetic analysis

Total DNA was isolated from individual specimens using a Chelex-100 with Proteinase-K solution. Forward primer 28sy (5′-CTA ACC AGG ATT CCC TCA GTA ACG GCG AGT-3′) and reverse primer 28sz (5′-AGA CTC CTT GGT CCG TGT TTC AAG AC-3′) (Hillis and Dixon 1991) were used to amplify partial 28S rDNA, and 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 et al. 1992) were used to amplify partial cox1 mtDNA gene. Polymerase chain reactions were performed in a total volume of 20 μl (11.5 μl H2O, 2.5 μl Taq buffer, 2 μl dNTP at a concentration of 10 pM, 0.5 μl of each primer at a concentration of 10 pM, 1 μl of Syntol Taq polymerase, 1 μl of the DNA template). The thermal cycler parameters were as follows: initial denaturation at 95°C (3 min); denaturation 20 s, 95°C; annealing 20 s at 62°C for 28sy/28sz primers, elongation 120 s at 72°C. For JB3/JB4.5 primers, annealing 20 s at 48.9°C and elongation 50 s at 72°C were performed. Final extension 5 min at 72°C for both primer pairs with 35 cycles of polymerase chain reaction was used. All amplicons were sequenced using the equipment of the Research Park of St. Petersburg State University (Centre for Molecular and Cell Technologies). Sequences from both forward and reverse primers were assembled using Chromas Pro v. 1.7.4 (Technelysium Pty Ltd).

To assess the phylogenetic position of species under consideration, Maximum Likelihood (ML) and Bayesian Inference (BI) analyses were performed on the 28S rDNA and cox1 gene dataset (Table 1). The sequences were downloaded using custom script based on ‘ape’ v. 5.0 package (Paradis and Schliep 2019) in ‘R’ programming language (R core team 2022).

The general alignment of partial 28S rDNA and cox1 gene sequences were generated with ‘MAFFT’ v7.520 (Katoh and Standley 2013) as implemented in Conda environment (Anaconda Software Distribution 2020) and then checked manually in SeaView v. 4 software (Gouy et al. 2010). The final length of the alignment was 876 bp for partial 28S rDNA sequence and 312 bp for cox1 gene. The evolutionary model for the phylogenetic analysis was estimated with MrModeltest v. 2.4 (Nylander 2004). The best fitted model was GTR + G + I in all cases. Maximum Likelihood analysis was made with RaxML-HPC2 on XSEDE at CIPRES portal (Miller et al. 2010) with 1000 pseudoreplicates. Bayesian analysis was performed using MrBayes v. 3.2.7a at CIPRES portal for 15,000,000 generations. The quality of the chains was estimated using built-in MrBayes tools performed on the local workstation. Based on the estimates, the first 5,000 generations were discarded for burn-in for 28S rDNA and cox1 mtDNA datasets. We included Paragonimus westermani (Rudolphi, 1819) (Plagiorchiida, Troglotrematidae) as an outgroup in our analysis according to the recent phylogenetic reconstructions (Perez-Ponce de Leon and Hernandez-Mena 2019).

Pairwise distances (p-distances) were calculated based on partial cox1 gene sequences with MEGA11 software (Tamura et al. 2021) under standard parameters. Heatmap was made with ‘ComplexHeatmap’ library (Gu, 2022) in ‘R’ programming language using native clusterization algorithm.

Results

Molecular phylogenetic analysis

Phylogenetic trees of Plagiorchis spp. based on the 28S rDNA sequences were different in ML and BI analyses. Therefore, we describe the results of these two analyses separately. In ML analysis (Figure 1A), two sequences of Plagiorchis mordovii are unresolved in relation to a poorly resolved clade of P. muelleri. Plagiorchis vespertilionis forms a sister clade to P. mordovii and P. muelleri. Plagiorchis koreanus occupies a basal position to P. mordovii, P. muelleri, and P. vespertilionis. Other Plagiorchis spp. branch in the following order: P. neomidis, P. maculosus, P. elegans.

Figure 1. Results of ML (A) and BI (B) analyses based on partial 28S rDNA sequences. Supports below 0.5 not shown. Newly obtained sequences are underlined.

In BI analysis (Figure 1B), P. mordovii and P. muelleri form two clades. Sequences of P. muelleri have a high nodal support, while those of P. mordovii have an extremely low nodal support. The branching node of these clades has a relatively high but still unreliable support. Plagiorchis vespertilionis appears to be the sister clade to these two species. Plagiorchis koreanus branches as a sister clade to all the species just mentioned. Other species of the genus examined in our study form a single clade, in which P. neomidis occupies a basal position.

The branching order of the ML and BI trees based on cox1 mtDNA sequences was the same (Figures 1 and 2). The sequences of P. mordovii form a clade that is sister to P. muelleri, with a high nodal support in BI analysis. Plagiorchis mordovii and P. muelleri are a sister clade to the clade formed by P. neomidis and P. koreanus. Plagiorchis elegans and P. maculosus occupy the basal position to all the other species under study.

Figure 2. Resuts of molecular phylogenetic analysis based on partal sequence of coxI mtDNA. Supports (ML/BI) below 0.5 not shown. Newly obtained sequences are underlined.

No differences were found between two generated sequences of P. mordovii; p-distance equal to 0.01 was observed between newly obtained sequences of P. mordovii, OR515269 and OR515270 (Figure 3). Distances between P. mordovii and P. muelleri are 0.09 (0.09–0.1), P. mordovii and P. maculosus are 0.16 (0.16–0.16), P. mordovii and P. elegans are 0.14 (0.14–0.14), P. mordovii and P. koreanus are 0.12 (0.12–0.13), and P. mordovii and P. neomidis are 0.14 (0.14–0.14). Differences between sequences of P. mordovii and P. westermani are equal to 0.25 (0.23–0.26). Thus, interspecific differences were about 0.12 in average, and all intraspecific ones were equal or less than 0.03 (all values are shown in cells on Figure 3).

Figure 3. Matrix of pairwise distances calculated on partial sequence of coxI mtDNA converted to a heatmap.a

Systematics and morphological characteristics of Plagiorchis spp.

Superfamily Microphalloidea Ward, 1901

Family Plagiorchiidae Lühe, 1901

Genus Plagiorchis Lühe, 1899

Plagiorchis vespertilionis (Müller, 1784) (Figure 4A)

Figure 4. Plagiorchis vespertilionis from Myotis daubentonii, whole view (A); Plagiorchis koreanus from Myotis daubentonii, whole view (B); Plagiorchis koreanus from Nyctalus noctula, whole view (C). Scale bars 0.5 mm.

Host: Myotis daubentonii.

Geographical locality: Shiryaevo village, Samarskaya Luka National Park (Samara region, Russia).

Availability: GenBank No OR514613.

Accession numbers in collection of IEVB RAS: No 332–335.

Trematodes P. vespertilionis from different individuals of M. daubentonii were morphologically similar. Characteristic features distinguishing this species from congeneric species were always present. At the same time, different specimens of P. vespertilionis showed some variability in the size of the body and the organs (Table 2).

Table 2. Measurements of trematode species under consideration

Note: mean values are given in parentheses; OS: oral sucker, VS:ventral sucker.

Plagiorchis koreanus Ogata, 1938 (Figure 4B, C)

Hosts: Myotis daubentonii, Nyctalus noctula.

Geographical localities: Shiryaevo village, Samarskaya Luka National Park (Samara region, Russia), Smolny village, Smolny National Park (Republic of Mordovia, Russia).

Availability: GenBank No OR514617, OR514618, OR515274.

Accession numbers in collection of IEVB RAS: No 373–376, 2074–2077.

Specimens of Plagiorchis koreanus from M. daubentonii and N. noctula were morphologically similar (Figure 4B, C). The distinctive features of this species were always present. Variability in the size of the body and the organs was noted in specimens of P. koreanus recovered from bats belonging to different species, from different bat individuals belonging to the same species, and even from one and the same bat individual. For instance, the specimens ex M. daubentonii were much larger than those from N. noctula (Table 2). The ratio of body length to width was relatively constant in specimens of P. koreanus from different host species. Howerver, this feature varied in specimens from bat individuals of the same host species (Table 2). The size of the oral and the ventral sucker varied in specimens of P. koreanus from different bat species and from different individuals of the same bat species (Table 2). The ratio of the oral sucker width to the ventral sucker width (OS/VS) was relatively constant in specimens of P. koreanus from different host species. At the same time, this feature varied in specimens from conspecific bat individuals (Table 2). The pharynx size varied in the specimens of P. koreanus ex M. daubentonii and ex N. noctula. The esophagus length was greater in specimens from M. daubentonii than in specimens from N. noctula (Table 2). The variability of the distance between the oral and the ventral sucker (distance from OS to VS) and the distance between the ventral sucker and the ovary (distance from ovary to VS) is more pronounced in specimens from bats of different species than in specimens from bat individuals of the same species.

The size of testes, ovaries, and the cirrus sac was variable in specimens of P. koreanus from different host species and from different individuals of the same host species. The reproductive organs and the eggs were significantly larger in specimens ex M. daubentonii (Figure 4B, C and Table 2).

Plagiorchis muelleri Tkach & Sharpilo, 1990 (Figure 5)

Figure 5. Plagiorchis muelleri from Eptesicus nilssoni, whole view, (A, B, C). Scale bar 0.5 mm.

Host: Eptesicus nilssoni Keyserling & Blasius, 1839.

Geographical locality: Shiryaevo village, Samarskaya Luka National Park (Samara region, Russia).

Availability: GenBank No OR514614, OR515271–OR515273.

Accession numbers in collection of IEVB RAS: No 318–321.

All specimens of P. muelleri from E. nilssoni were morphologically similar. The only variable character was the location of the anterior border of vitelline fields (Figure 5). In some specimens, vitelline fields could reach the level of the distal part of the cirrus sac but never extended beyond it (Figure 5A). In some other specimens, vitelline fields reached only the posterior margin of the ventral sucker (Figure 5B). In most cases, however, the anterior edge of vitelline fields was located at the level of the ventral sucker (Figure 5C). A slight variability in the body shape was revealed (Figure 5). The size of the body and the organs was variable (Table 2). The characteristic features of P. muelleri were always present.

Plagiorchis mordovii Shaldybin, 1958 (Figure 6)

Figure 6. Plagiorchis mordovii from Myotis brandtii, whole view, Samara region (A); Plagiorchis mordovii from Myotis brandtii, whole view, Mordovia (B). Scale bar 0.5 mm.

Host: Myotis brandtii.

Geographical locality: Shiryaevo village, Samarskaya Luka (Samara region, Russia), Smolny village, Smolny National Park (Republic of Mordovia, Russia).

Availability: GenBank No OR514615, OR514616, OR515268–OR515270.

Accession numbers in collection of IEVB RAS: No 392–396, 2069–2073.

General description (based on 30 mature specimens). Body elongate-oval, slightly tapering towards anterior end. Posterior body wide and rounded. Anterior body part covered with spines gradually disappearing towards posterior body part. Oral sucker rounded, subterminal. Ventral sucker located at border of first and second thirds of body, approximately equal to or somewhat smaller than oral sucker. Esophagus absent or poorly expressed. Intestinal branches reach hind body. Testes rounded or oval, entire, lying symmetrically in midbody part, approximately at same level or one testis slightly in front of other. Cirrus sac located medially in area of ventral sucker and ovary, making S-shaped bend. Approximately 1/2 or 1/3 of cirrus sac length overlapped by ventral sucker. Proximal end of cirrus sac sometimes reaching posterior margin of ovary or anterior margin of testis. Genital pore medial, located anteriorly to ventral sucker. Ovary rounded or oval, located submedially, behind ventral sucker, at some distance from it. Ovary always smaller than ventral sucker. Vitelline fields consisting of small irregular follicles, located laterally. Posteriorly, vitelline follicles arranged more densely. Anterior margin of vitellarium located approximately in middle of distance between suckers. Posterior edge of vitelline fields never reaching hindbody. Vitelline fields do not merge, but single follicles in anterior body part and at level of testes may reach body midline. Genital pore medial, at anterior margin of ventral sucker. Uterus passes between testes and fills all space behind them. Metraterm well-defined, beginning at ovary level.

Remarks. Specimens of P. mordovii ex M. brandtii from various study areas were morphologically similar. Only slight differences in the body shape were noted (Figure 6A, B). There was also some variability in the length of vitelline fields. In most specimens, the anterior edge of the vitellarium was located approximately midway between the oral and the ventral sucker, usually closer to the latter (Figure 6A). At the same time, in some specimens the anterior edge of the vitellarium was located closer to the oral sucker, though it did not reach the level of the posterior edge of the pharynx (Figure 6B). In addition, in some trematode specimens, some of the vitelline follicles on dorsal body side between suckers could reach the body midline (Figure 6B). We also noted variability in the location of the testes, which could be located approximately at the same level (Figure 6B) or somewhat diagonally to each other (Figure 6A).

The specimens of P. mordovii differed in the size of the body and organs (Table 2). The largest specimens were found in M. brandtii from the Samara region. Specimens from bats in Mordovia were smaller (Table 2). Variability in body size and the ratio of body length to width was less pronounced in specimens from bats from the same study area. The size of the suckers and the pharynx in specimens of P. mordovii from different study areas was approximately the same, and so was the ratio of the oral sucker width to the ventral sucker width. The differences in these features were more pronounced in specimens from bats of the same region. A strong variability was found in the distance between the ventral sucker and the ovary. The distance between the oral and the ventral sucker was less variable (Table 2). The size of the testes, the ovaries, and the cirrus sac varied greatly in specimens from different study areas. The characters of specimens from bats of the same study area were less variable. The largest reproductive organs were found in specimens of P. mordovii from bats in the Samara region (Table 2). Egg size was relatively constant regardless of the study area.

Discussion

In this study, we analyzed morphological features and morphometric characteristics of four Plagiorchis spp. from bats in the Samara region and Mordovia (Russia) and obtained new molecular phylogenetic data on them. The combined use of molecular and morphological methods made it possible to perform a reliable identification of Plagiorchis koreanus, P. mordovii, P. muelleri, and P. vespertilionis.

Species identity of the adult trematodes described in our study was supported by molecular phylogenetic analysis. In phylogenetic reconstructions, the species clustered in accordance with their morphological identification based on live specimens. Using partial sequences of 28S rDNA and cox1 mtDNA, we showed that P. mordovii was a separate lineage within Plagiorchis spp. considered in our study. Pairwise distance estimation supported this hypothesis. The pairwise distances calculated on partial cox1 mtDNA sequences of P. mordovii were equal or less than 0.01, and about 0.13 in average between P. mordovii and other specimens. Thus, these values correspond to intraspecific and interspecific, respectively.

In order to determine phylogenetic connections of P. mordovii with other Plagiorchis spp., we considered, in addition to the newly obtained sequences, several sequences available in public databases. In general, our phylogenetic tree based on 28S rDNA sequences was in good agreement with the pioneering study of Tkach et al. (2000b) based on ITS2 region sequences of nuclear DNA. However, Tkach et al. (2000b) studied only species of the P. vespertilionis group (P. vespertilionis, P. muelleri, P. koreanus). Species distant from the P. vespertilionis group were studied by Kudlai et al. (2021). The general topology of the BI tree in our study is in full agreement with their BI tree based on 28S rDNA sequences. The fact that ML and BI trees were mismatched in our study may be explained by a relatively short alignment.

We provided a complete description of Plagiorchis mordovii ex M. brandtii with morphometric characteristics and confirmed the validity and the generic affiliation of this species. We reported P. koreanus ex M. daubentonii and N. noctula for the first time.

In general, the specimens of all Plagiorchis spp. examined in our study corresponded very well to the previous descriptions of these species (Tables 2, S1, S2, S3, S4). The characteristic features of the species could always be clearly seen, such as the position of the anterior edge of the vitelline fields, the shape and position of the cirrus sac, the oral and the ventral sucker width ratio, the body length and width ratio, the distance between the ventral sucker and the ovary, and the position of the testes.

At the same time, we noted some differences in the topology of inner organs and the morphometric characteristics. The specimens showed a broad morphological variability. This concerns specimens from various host species and from the same host species, as well as specimens from the same and from different study area and habitats. The body length and width and the size of the reproductive organs varied broadly. The body length and width ratio, the width ratio of oral sucker to ventral sucker, the distance between oral and ventral sucker, and the distance between ventral sucker and ovary were also variable. Morphological variability in the size of the pharynx, the oral, and the ventral sucker was less pronounced. The egg size was relatively stable. Comparison of our specimens of P. mordovii with the previous descriptions by Shaldybin in Skrjabin and Antipin (1958) and Zdzitowiecki (1970) showed that the main morphological and morphometric characteristics were very similar, with the exception of the body size and the size of the cirrus sac. Our specimens were larger and had a longer cirrus sac (Tables 2, S1). In the case of P. muelleri, the position of the anterior edge of vitelline fields in our specimens differed slightly from the earlier descriptions (Tkach and Sharpilo 1990; Tkach et al. 2000b). The only exception in P. koreanus morphology was the length of cirrus sac, which was longer in our specimens than in those described by other authors (Tables 2, S4).

Plagiorchis spp. are generally characterized by a significant morphological and morphometric variability (Krasnolobova 1987; Tkach and Sharpilo 1990; Sharpilo and Tkach 1992; Tkach et al. 2000b; Kirillov et al. 2012a; Kirillov and Kirillova 2010). Tkach et al. (2000b) have noted that the specimens of P. koreanus, P. muelleri, and P. vespertilionis are morphologically similar. This similarity has sometimes led to confusion. For instance, the species identified as P. vespertilionis in Groschaft and Tenora (1973), Meszaros and Mas-Coma (1980), and Alvarez et al. (1991) belong, judging by the figures in those works, to P. muelleri. Plagiorchis koreanus has been mistaken for P. vespertilionis Matsaberidze & Khotenovsky (1967), although the figure shows all the morphological features of P. koreanus.

It was often difficult to distinguish P. koreanus and P. muelleri. Plagiorchis vespertilionis is easily distinguished from P. koreanus and P. muelleri by the position of the anterior border of the vitellarium, the shape of the cirrus sac and its position, and by the body length and width ratio and the distance between the ventral sucker and the ovary, but P. koreanus and P. muelleri are similar in these characteristics. The main differences between these two species are the oral and the ventral sucker ratio (in P. koreanus, the oral sucker is always larger than the oral one) and the presence of the esophagus (in P. koreanus, the esophagus is always well expressed). In addition, in P. muelleri, the vitelline fields always merge dorsally behind the ovary and testes, while in our specimens of P. koreanus the vitelline fields did not merge.

This means that morphometric features such as the body size, the size of the oral and the ventral sucker and reproductive organs, and the distance between the oral and the ventral sucker are insufficient for identifying P. koreanus, P. muelleri, and P. vespertilionis. The values of these parameters overlapped in some specimens in our study. Tkach et al. (2000b) have indicated that the most reliable morphometric features of these species were the body length to width ratio, the oral sucker to ventral sucker width ratio, and the distance between the ventral sucker and the ovary. We came to the same conclusion.

Plagiorchis mordovii is similar in some morphological and morphometric features to P. elegans, such as the absence of the esophagus, the position of vitelline fields, the body length, and the size of testes and eggs. This may also lead to confusion. For example, Demidova and Vekhnik (2004) identified the specimens of P. mordovii from M. brandtii as P. elegans, although the drawing clearly shows the morphological features of P. mordovii.

We have previously found Plagiorchis elegans in P. auritus from the Samara region and in V. murinus and N. noctula from Mordovia (Kirillova et al. 2007; Kirillov et al. 2012a, 2012b; Kirillova et al. 2023). When identifying P. mordovii and P. elegans, the following distinguishing features should be taken into account:

1. Body shape. In P. mordovii, the body is more oval than elongated, while in P. elegans the body is elongated-oval.

2. Position of the testes. In P. mordovii, the testes usually lie symmetrically. Sometimes one testis is slightly shifted relative to the other but not by more than half of its length. In P. elegans, the testes always lie diagonally.

3. Position of the uterus. In P. mordovii, the uterus does not form an S-shaped bend between the testes, as it does in P. elegans.

4. Shape of the cirrus sac and its position in relation to the ventral sucker and the ovary. In P. mordovii, the cirrus sac makes an S-shaped bend near the ventral sucker and the ovary, while in P. elegans it makes a C-shaped bend near the ventral sucker.

The distinctive morphometric characteristics of P. mordovii and P. elegans are showed in Table 3.

Table 3. Comparison of morphometric characteristics of P. mordovii and P. elegans

Note: mean values are given in parentheses; OS: oral sucker, VS: ventral sucker.

Plagiorchis mordovii differs quite well from other Plagiorchis spp. parasitizing bats in the following features: the body shape, the body length and width ratio, the position of the anterior edge of the vitelline fields, the testes, the uterus, and the cirrus sac.

Based on the analysis of the morphological and morphometric features of adult Plagiorchis spp. parasitizing bats, we propose a key for their identification.

Key to Plagiorchis species parasitizing European bats

1(6) Anterior edge of vitelline fields between oral and ventral sucker.

2(4) Testes lie diagonally.

3(2) Ovary always larger than ventral sucker. Cirrus sac C-curved around ventral sucker. Uterus forms an S-shaped bend between the testes. ………….….….….….….….….….….…. Plagiorchis elegans (Rudolphi, 1802).

4(2) Testes lie symmetrically, or one testis is slightly shifted relative to the other but not more than by half of its length.

5(4) Ovary always smaller than ventral sucker. Cirrus sac S-curved in the area of ventral sucker and ovary. Uterus does not form an S-shaped bend between the testes. ……… Plagiorchis mordovii (Shaldybin, 1958).

6(11) Anterior edge of vitelline fields at level of ventral sucker.

7(9) Oral sucker larger than ventral sucker.

8(7) Esophagus always well defined. …….….….….….… Plagiorchis koreanus Ogata, 1938.

9(7) Oral sucker of the same size as ventral sucker.

10(9) Esophagus absent or poorly expressed. Metraterm begins midway between ventral sucker and ovary. Body length to width ratio is 3.42–4.83:1. ……………………………………… Plagiorchis muelleri Tkach & Sharpilo, 1990.

11(6) Anterior edge of vitelline fields below level of ventral sucker.

12(11) Oral sucker smaller than ventral sucker. Metraterm begins at ovary level. Body length to width ratio is 5.21–9.63:1. …….….….….….….….….….….….….….….….….…..….‥. Plagiorchis vespertilionis (Müller, 1784).

An analysis of our results and the literature data showed that Plagiorchis spp. have a certain host specificity. Thus, Plagiorchis vespertilionis occurs predominantly in M. daubentonii. It has been reported from other bats (Soltys 1959; Skvortsov 1972; Sharpilo and Iskova 1989; Tkach et al. 2000b; Shimalov et al. 2011; Kirillov et al. 2012a, 2012b, 2015; Sumer and Yildirimhan, 2017, 2018, 2019; Lebedeva et al. 2020; Shimalov 2021; Kirillova et al. 2023), but, in our opinion, these findings require verification. The authors might have dealt with closely related P. muelleri and P. koreanus, which are common in bats.

Plagiorchis mordovii has been found in M. dasycneme, M. brandtii, and V. murinus (Skrjabin and Antipin 1958; Zdzitowiecki 1970; Demidova and Vekhnik 2004; Kirillov et al. 2012a, 2012b; Kirillova et al. 2023). Plagiorchis muelleri and Plagiorchis koreanus parasitize many European bats (Shtrom 1940; Tkach and Sharpilo 1990; Sharpilo and Tkach 1992; Tkach et al. 2000b; Demidova and Vekhnik 2004; Kirillova et al. 2007, 2023; Kirillov et al. 2012a, 2012b; Sumer and Yildirimhan 2017, 2018). Plagiorchis elegans is uncommon in bats and has been occasionally found only in M. alcathoe, M. daubentonii, P. auritus, V. murinus, and N. noctula (Kirillov et al. 2012a, 2012b; Kirillova and Kirillov 2017; Sumer and Yildirimhan 2019; Kirillova et al. 2023).

Thus, a combined use of molecular and morphological methods in our study made it possible to reliably identify closely related Plagiorchis spp.: P. vespertilionis, P. koreanus, P. mordovii, and P. muelleri. We provided a complete morphological description of Plagiorchis mordovii for the first time and confirm its validity and generic affiliation.