Introduction
The family Opecoelidae Ozaki, 1925 is a large group of trematodes parasitic in marine and freshwater fish. The modern taxonomic model of this family, based on morphological (Cribb, Reference Cribb, Jones, Bray and Gibson2005; Bakhoum et al., Reference Bakhoum2017) and genetic data (Bray et al., Reference Bray2016; Martin et al., Reference Martin2018b, Reference Martin, Cutmore and Cribbc), distributes opecoelids between six subfamilies: Opecoelinae, Helicometrinae, Opecoelininae, Opistholebetinae, Stenakrinae and Plagioporinae s. lato. Plagioporinae s. lato is the most problematic group of opecoelids from the taxonomic point of view, as phylogenetic reconstructions demonstrate the polyphyly of this subfamily (Andres et al., Reference Andres, Pulis and Overstreet2014; Shedko et al., Reference Shedko, Sokolov and Atopkin2015; Bray et al., Reference Bray2016; Fayton and Andres, Reference Fayton and Andres2016; Faltýnková et al., Reference Faltýnková, Klimpel, Kuhn and Mehlhor2017; Fayton et al., Reference Fayton2017; Martin et al., Reference Martin, Cutmore and Cribb2017, Reference Martin2018b; Rima et al., Reference Rima2017; Fayton et al., Reference Fayton2018).
In this study, we report on two opecoeline species, Pseudopecoelus cf. vulgaris and Anomalotrema koiae Gibson & Bray, 1984, from fish collected in the boreal waters of the North Pacific. Previously, A. koiae was recorded as a parasite only on North Atlantic fish (e.g. Gibson, Reference Gibson, Margolis and Kabata1996), and this is the second record of Pseudopecoelus cf. vulgaris in the North Pacific (see Machida and Araki, Reference Machida and Araki1992). We detail the phylogeny of the family Opecoelidae, taking into account new partial sequences of 28S rDNA of these two species and six other previously unsequenced representatives of the family.
Materials and methods
Specimen collection
The trematodes Pseudopecoelus cf. vulgaris and A. koiae were collected during a parasitological examination of North Pacific fishes Lycodes brunneofasciatus Suvorov, 1935, Microankathus fedorovi (Mandrytsa, 1991) and Sebastes sp. (table 1). A total of 15 specimens of Lycodes brunneofasciatus (length 21.0–52.5 cm, weight 135–910 g), 30 specimens of M. fedorovi (length 7.5–17.0 cm, weight 19.3–45.0 g), and nine specimens of Sebastes sp. (length 30.2–45.0 cm, weight 415–1185 g) were examined in this research. All the specimens were caught by the fishing vessel Anatoly Torchinov, when it was fishing for Pleurogrammus monopterygius (Pallas, 1810) during 16–25 March 2017 at depths of 150–400 m in the environs of Simushir Island. The fish nomenclature was adopted following Voskoboinikova (Reference Voskoboinikova2015) and Froese and Pauly (Reference Froese and Pauly2017).
List of newly obtained sequences.
Additionally, novel genetic data are provided for six other opecoelids, namely adult Sphaerostoma bramae (Müller, 1776), Nicolla skrjabini (Iwanitzky, 1928), Discoverytrema gibsoni Zdzitowiecki, 1990, Discoverytrema markowskii Gibson, 1976 and Macvicaria muraenolepidis Zdzitowiecki, 1990, and Podocotyle atomon (Rudolphi, 1802) cercariae (table 1).
The adult worms collected for morphological study were fixed in 70% ethanol at room temperature under a glass cover without additional pressure and stained with acetocarmine. Species identification was performed following Gibson and Bray (Reference Gibson and Bray1984), Bykhovskaya-Pavlovskaya and Kulakova (Reference Bykhovskaya-Pavlovskaya, Kulakova and Bauer1987), Sokolov and Gordeev (Reference Sokolov and Gordeev2013), and Blend et al. (Reference Blend2017). The identification of the P. atomon cercariae was carried out according to the descriptions given in Hunninen and Cable (Reference Hunninen and Cable1943) and Chubrik (Reference Chubrik and Polanski1966). According to modern data, in littoral molluscs on the north-western coast of the White Sea, there is only one species of cercariae: P. atomon (Levakin et al., Reference Levakin, Nikolaev and Galaktionov2013). In the White Sea, another species of the genus Podocotyle Dujardin, 1845 is also noted: P. reflexa (Creplin, 1825) (see Shulman and Shulman-Albova, Reference Shulman and Shulman-Albova1953). However, the morphology of cercariae of this species differs sharply from those of the larvae of P. atomon: there are three pairs of penetration glands in P. atomon, and four in P. reflexa (Koie, 1981). All the lengths in morphological descriptions are in μm unless otherwise noted. Specimens destined for molecular analysis (table 1) were fixed in 96% ethanol and stored at 4°C.
DNA extraction, amplification and sequencing, and phylogenetic analysis
In order to obtain 28S rDNA sequence, total DNA was isolated using the ZymoBead Genomic DNA Kit (http://www.zymoresearch.com). Only single trematode specimens were used for each DNA extraction. The DNA fragment of c. 1200 bp localized at the 5′ end of 28 rDNA was amplified using the BIO-RAD C1000 Thermal Cycler. Polymerase chain reactions (PCR) 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 Taq polymerase (‘Syntol’), 1 μl of the DNA template).
The trematode-specific forward primer LSU-5 (5′-TAG GTC GAC CCG CTG AAY TTA AGC A-3′) and reverse primer 1500R (5′-GCT ATC CTG AGG GAA ACT TCG-3′) were used. GenBank numbers of sequences used in the analysis are provided in table 2. The thermal cycle parameters were as follows: initial denaturation at 95°C (3 minutes); 35 cycles of 20 s at 95°C; 20 s at 56°C; 120 s at 72°C; 5 minutes at 72°C for the final extension. Amplicons were purified using the Cleanup Mini Purification Kit (Eurogene). All amplicons were sequenced directly using equipment belonging to the Research Park of Saint Petersburg State University (Centre for Molecular and Cell Technologies). Sequences from both forward and reverse primers were assembled in Chromas Pro 1.7.4. Each sequence obtained was amplified from primers whose landing sites are at a great distance from each other. Consequently, the sequences obtained from single instances of the worms were manually corrected, and the sequences obtained were consensual. The voucher specimens of the studied species were deposited in the specimen collection at the Department of Invertebrate Zoology at Saint Petersburg State University.
List of species incorporated into molecular analysis.
The newly obtained sequences were included in a general alignment (table 1). Firstly, the sequences were automatically aligned using the MUSCLE algorithm (Edgar, Reference Edgar2004), as implemented in SeaView 4.0 (Gouy et al., Reference Gouy, Guindon and Gascuel2010). Then the alignment was verified manually. The phylogenetic analysis was performed using the maximum likelihood (ML) method, as implemented in the RaxML program (Stamatakis, Reference Stamatakis2006) at CIPRES Science Gateway (www.phylo.org) (Miller et al., Reference Miller, Pfeiffer and Schwartz2010). About 1000 sites were selected for the analysis. The stability of the clades was assessed using a non-parametric bootstrap with 1000 pseudoreplicates. All estimations in RaxML were calculated using default parameters.
Bayesian inference (BI) analysis was performed using MrBayes 3.1.2, a general time reversible (GTR) model with gamma correction for intersite rate variation (eight categories), and the covarion model. Trees were run as two separate chains (default heating parameters) for 15 million generations, at which point they had ceased converging. The quality of chains was estimated using built-in MrBayes tools and, additionally, the Tracer 1.6 package (Rambaut et al., Reference Rambaut2014). Based on the estimates using Tracer, 250,000 generations were discarded for burn-in (the relative burn-in parameter was switched off). The names of the clades are given following Martin et al. (Reference Martin2018b, Reference Martin, Cutmore and Cribbc).
Results
Systematics
Family Opecoelidae Ozaki, 1925
Subfamily Opecoelinae Ozaki, 1925
Genus Pseudopecoelus von Wicklen, 1946
Pseudopecoelus cf. vulgaris (fig. 1A)
Description
(Based on five gravid specimens from L. brunneofasciatus). Body elongate-oval, flattened dorso-ventrally, 1.90–3.81 × 0.52–0.95 mm. Tegument unarmed. Oral sucker subspherical, unspecialized, 186–285 × 174–242. Ventral sucker slightly protuberant, subellipsoidal, unspecialized, 248–446 × 397–521; aperture surrounded by lip-like folds of tegument. Ratio of oral sucker width to ventral sucker width 1 : 2.10–2.29. Forebody 19.5–30.1% of body length. Mouth subterminal. Prepharynx not visible. Pharynx muscular, 153–199 × 92–135; oesophagus 167–360. Intestinal bifurcation short distance anterior to ventral sucker. Caeca terminate blindly near posterior extremity. Testes two, in third quarter of body, median, tandem, contiguous, lobated; anterior testis 186–496 × 310–546, posterior testis 248–459 × 298–583. Post-testicular region 533–980. Cirrus-sac absent. Seminal vesicle tubular, convoluted, free in parenchyma, extending some distance into hindbody. Pars prostatica surrounded by prostatic gland-cells; ejaculatory duct thick-walled, short. Genital pore at level of pharynx, sinistro-submedian. Ovary 4-lobed, median, pretesticular, contiguous with anterior testis, 174–273 × 260–397. Oviduct arising from anteromedial lobe of ovary. Laurer's canal opening on dorsal side of body between sinistral caecum and sinistral body margin, at level of ovary. Seminal receptacle uterine. Mehlis' gland distinct. Uterus preovarian, intercaecal. Metraterm not visible. Vitellarium follicular; follicles in two longitudinal lateral fields extending from ventral sucker region or intestinal bifurcation level to posterior extremity of body, frequently one field slightly longer than other. Vitelline fields overlapping caeca ventrally and confluent in post-testicular region on ventral side of body. Dorsal part of each field covers caecum only in ventral sucker and inter-gonadal areas; in post-testicular region divided into two rows: extra-caecal and submedian intra-caecal. Eggs numerous, oval, operculate, with small knob at anopercular pole, 79–82 × 41–47. Excretory bladder tubular, reaching to ovary; pore terminal.
(A) Pseudopecoelus cf. vulgaris, ventral view, and (B) Anomalotrema koiae, dorsal view (the basal part of the pedunculate ventral sucker is indicated by a dotted line). Scale bars: A, 1 mm; B, 0.5 mm.
Taxonomic summary
Host. Lycodes brunneofasciatus (Perciformes: Zoarcidae) and Sebastes sp. (Scorpeniformes: Sebascidae).
Locality. North Pacific in the environs of Simushir Island, 46°28′S, 150°59′E, depth 160 m.
Date of collection. 20 March 2017.
Site of infection. Intestine.
Prevalence and intensity. L. brunneofasciatus – 40% (n = 15), 3–10 individuals; Sebastes sp. – 55.6% (n = 9), 1 individual.
Specimens deposited. Mount no. 14266 in the Museum of Helminthological Collections at the Centre for Parasitology of the A.N. Severtsov Institute of Ecology and Evolution (IPEE RAS), Moscow, Russia.
Systematics
Family Opecoelidae Ozaki, 1925
Subfamily Opecoelinae Ozaki, 1925
Genus Anomalotrema Zhukov, 1957
Anomalotrema koiae Gibson & Bray, 1984 (fig. 1B)
Description
(Based on two gravid specimens from M. fedorovi). Body elongate, flattened dorso-ventrally, 1.60–1.86 × 0.37–0.5 mm. Tegument unarmed. Oral sucker subellipsoidal, unspecialized, 122 × 147–171. Ventral sucker pedunculate, with two sublateral muscular lobes on anterior margin and single median lobe on posterior margin, 322–360 × 260–285. Ratio of oral sucker width to ventral sucker width 1 : 1.67–1.77. Mouth subterminal. Prepharynx 52, muscular pharynx 92–122 × 110–159, oesophagus 183. Caeca extending dorso-laterally to posterior extremity of body and opening through separate ani, which lie ventrally to excretory pore. Testes two, in third quarter of body, median, tandem, contiguous entire, oval; anterior testis 174–186 × 236–298, posterior testis 217–233 × 248–273. Post-testicular region 322–446. Vas deferens absent; vasa efferentia joined directly to internal seminal vesicle. Cirrus-sac membranous, containing convoluted internal seminal vesicle, tubular pars prostatica and ejaculatory duct. Distal region of seminal vesicle surrounded by prostatic gland-cells. Genital pore at level of oesophagus, sinistro-submedian. Ovary three-lobed median, pretesticular, contiguous with anterior testis 159 × 190–226. Laurer's canal opening dorsally to sinistral caecum at level of ovary. Seminal receptacle uterine. Mehlis' gland distinct. Uterus preovarian, intercaecal. Metraterm muscular. Vitellarium follicular in two longitudinal lateral fields; fields extending from proximal part of external seminal vesicle to posterior extremity of body, interrupted at level of gonads and confluent in post-testicular region. Vitelline reservoir dorsal to ovary. Eggs numerous, 64–66 in length (deformed in Canada balsam). Excretory bladder tubular, extending to ovarian zone; pore terminal.
Taxonomic summary
Host. Microancathus fedorovi (Scorpeniformes: Cyclopteridae).
Locality. North Pacific in the environs of Simushir Island, 47°13′S, 152°24′E, depth 210 m.
Date of collection. 25 March 2017.
Site of infection. Intestine.
Prevalence and intensity. 6.7% (n = 30), 1–3 individuals.
Specimens deposited. Mounts no. 14264 and 14265 in the Museum of Helminthological Collections at the Centre for Parasitology of the A.N. Severtsov Institute of Ecology and Evolution (IPEE RAS), Moscow, Russia.
Phylogenetic data
The BI and ML trees were similar terms of general topology (figs 2 & 3). All opecoelids were distributed between six well-supported clades – Helicometrinae, Opecoelinae, Opistholebetinae, ‘freshwater Plagioporinae’, ‘marine Plagioporinae B’ and ‘marine Plagioporinae C’, and also the paraphyletic group ‘deep-sea Plagioporinae’ and the poorly supported clade ‘Pedunculacetabulum inopinipugnus +Podocotyloides stenometra’ (fig. 2).
Phylogenetic tree of the Opecoelidae based on the analysis of 28S rDNA partial sequences using ML and BI algorithms; nodal numbers indicate bootstraps/posterior probabilities. The brachycladiid species Zalophotrema hepaticum, Oshmarinella macrorchis and Campula oblonga were used as outgroups. Clades with grey shading have different supports in the BI and ML trees and are shown in fig. 3.
Pseudopecoelus cf. vulgaris and A. koiae were included in the clade Opecoelinae (fig. 2). Three sequences of 28S rDNA for Pseudopecoelus cf. vulgaris (GenBank no. MH161433 and MH161434 ex L. brunneofasciatus, and MH161436 ex Sebastes sp.) were identical. The newly obtained sequence of 28S rDNA in our own specimen of A. koiae was identical to that of A. koiae GenBank no. KU320595. Other species examined in this study were distributed across the tree as follows: D. gibsoni and D. markowskii were sister species to each other in the clade Opecoelinae; S. bramae and N. skrjabini were in the clade ‘freshwater Plagioporinae’, M. muraenolepidis was in the clade ‘marine Plagioporinae C’, and P. atomon was in the group ‘deep-sea Plagioporinae’.
Pseudopecoelus cf. vulgaris is a well-supported sister taxon of Pseudopecoeloides tenuis Yamaguti, 1940 within the clade Opecoelinae, and the group they form combines with Opecoeloides spp. to constitute a well-supported terminal subclade. This subclade has a weakly supported sister relationship with A. koiae. The Discoverytrema spp. group shares a common ancestor with a marine opecoelines group, consisting of A. koiae, Pseudopecoelus cf. vulgaris, Pseudopecoeloides tenuis and Opecoeloides spp. (fig. 2).
Macvicaria muraenolepidis was combined with Macvicaria magellanica Laskowski, Jeżewski & Zdzitowiecki, 2013 with high support, within the clade ‘marine Plagioporinae C’. Group M. muraenolepidis + M. magellanica was combined with Choerodonicola renko Machida, 2014 and Choerodonicola arothokoros Martin, Cribb, Cutmore, & Huston, 2018 into a very well-supported subclade, which is sister to the also strongly supported subclade Podocotyloides parupenei + Trilobovarium parvvatis (fig. 2).
Podocotyle atomon was clustered with the deep-sea plagioporines Allopodocotyle margolisi Gibson, 1995, Buticulotrema thermichthysi Bray, Waeschenbach, Dyal, Littlewood & Morand, 2014 and Gaevskajatrema halosauropsi Bray & Campbell, 1996. However, the strict interrelation between P. atomon and these species remains unresolved (figs 2 & 3). The position of S. bramae and N. skrjabini within the ‘freshwater Plagioporinae’ clade was poorly resolved (figs 2 & 3).
Mismatches between phylogenetic trees inferred with the ML and BI algorithms. On the left are fragments of the Bayesian tree and on the right are fragments of the ML tree.
Discussion
The genus Pseudopecoelus von Wicklen, 1946 unites 39 species (Blend et al., Reference Blend2017). The specimens that we studied are undoubtedly conspecific with the Pseudopecoelus sp. collected by Machida and Araki (Reference Machida and Araki1992) from Careproctus roseofuscus Gilbert & Burke, 1912 in the Sea of Okhotsk. In turn, these parasites are most similar to Pseudopecoelus vulgaris (Manter, Reference Manter1934) in terms of the size of the body, ratio of suckers, presence of lip-like folds around the ventral sucker, position of the genital pore and the anterior edge of the vitelline fields, seminal vesicle and gonad shape, position of the gonads very close together and continuous (without discontinuity) arrangement of vitelline follicles opposite to the testes (see Blend et al., Reference Blend2017). Nevertheless, the egg length in Pseudopecoelus cf. vulgaris specimens (= Pseudopecoelus sp. Machida & Araki, 1992) is less than that of gravid P. vulgaris s. str.; 79–83 vs 90–127 μm (this study; Manter, Reference Manter1934, Reference Manter1954; Machida and Araki, Reference Machida and Araki1992). For very young individuals of P. vulgaris s. str. with a body length of up to 1.39 mm, eggs of a smaller length, 78 μm, are indicated by Manter (Reference Manter1934). The trematodes examined by us cannot be classified as very young, based on both the length of the body (1.90–3.81) and the presence of numerous eggs in the uterus. The closest to the Simushir Island area that P. vulgaris s. str. have been discovered is Tosa Bay, off the Pacific coast of southern Japan (Kuramochi, Reference Kuramochi, Fujita, Saito and Takeda2001). Thus, molecular data on P. vulgaris s. str. are required for the clarification of the taxonomic relationship between these two parasites.
The genus Anomalotrema Zhukov, Reference Zhukov1957 contains two species: A. putjatini Zhukov, 1957 (type species) and A. koiae (see Gibson and Bray, Reference Gibson and Bray1984; Gibson, Reference Gibson, Margolis and Kabata1996). Morphological differences between these species are manifested in the length of the body (2.38–7.38 mm vs 1.35–4.4 mm), shape of the ovary (indistinctly lobed or bell-shaped vs clearly 3–4 lobed) and sucker ratio (1 : 1.47–1.6 vs 1 : 1.8–2.4) (Zhukov, Reference Zhukov1957, Reference Zhukov1963; Gibson and Bray, Reference Gibson and Bray1984; Gibson, Reference Gibson, Margolis and Kabata1996). Until now, A. putjatini has been recorded from North Pacific scorpeniform and gadiform fish (Zhukov, Reference Zhukov1957, Reference Zhukov1963; Korotaeva, Reference Korotaeva, Skrjabin and Mamaev1968), and A. koiae from North Atlantic scorpeniform, gadiform and pleuronectiform fish (Gibson, Reference Gibson, Margolis and Kabata1996; Gaevskaya, Reference Gaevskaya2002). Our specimens correspond with the description of A. koiae in all features except the sucker ratio, and they occupy a position intermediate between A. koiae and A. putjatini in the indicated feature. However, given the identity of our sample of 28S rDNA sequences compared with those of A. koiae (see above), we consider them conspecific with this species.
The inclusion of Pseudopecoelus cf. vulgaris, A. koiae and Discoverytrema spp. in the clade Opecoelinae corresponds with the generally accepted point of view regarding the subfamilial affiliation of these trematodes (Zdzitowiecki, Reference Zdzitowiecki1990a; Gibson, Reference Gibson, Margolis and Kabata1996; Cribb, Reference Cribb, Jones, Bray and Gibson2005; Blend et al., Reference Blend2017). The molecular data we obtained support the morphologically based monophyly of the genus Discoverytrema Gibson, 1976 (see Zdzitowiecki, Reference Zdzitowiecki1990a). The node support between A. koiae and the group that unites the representatives of the genera Opecoeloides Odhner, 1928, Pseudopecoelus, Pseudopecoeloides Yamaguti, 1940 is too low to be convincing (fig. 2). In this regard, it is impossible to ascertain yet which group among the mentioned opecoelines is the sister branch to the genus Discoverytrema. At the same time, present analysis demonstrates the sister position of the Opecoeloides spp. group with respect to the Pseudopecoelus cf. vulgaris + Pseudopecoeloides tenuis group. Previously published reconstructions of phylogeny of the opecoelids, which did not include Pseudopecoelus cf. vulgaris, have supported the view that P. tenuis is the sister taxon to the Opecoeloides spp. group (e.g. Bray et al., Reference Bray2016; Rima et al., Reference Rima2017; Fayton et al., Reference Fayton2018; Martin et al., Reference Martin, Cutmore and Cribb2018c). Based on these data, Bray et al. (Reference Bray2016) hypothesized monophyly of opecoelines with a uroproct. It is important to note that a uroproct is characteristic of Pseudopecoeloides spp. and Opecoeloides spp., but not Pseudopecoelus spp. (e.g. Cribb, Reference Cribb, Jones, Bray and Gibson2005). Therefore, the data we have obtained do not support this hypothesis. It is difficult to identify reliable synapomorphies for the Opecoeloides spp. + (Pseudopecoelus cf. vulgaris + Pseudopecoeloides tenuis) subclade or the Pseudopecoelus cf. vulgaris + Pseudopecoeloides tenuis group, because of the limited number of opecoelines with a phylogenetic position verified using the molecular methods.
There are no clear morphological differences between the clades of the Plagioporinae sensu Cribb, Reference Cribb, Jones, Bray and Gibson2005: ‘freshwater Plagioporinae’ ‘marine Plagioporinae B’, ‘marine Plagioporinae C’, Opistholebetinae, ‘deep-sea Plagioporinae’, and ‘Pedunculacetabulum inopinipugnus + Podocotyloides stenometra’ (Bray et al., Reference Bray2016; Martin et al., Reference Martin2018b). The concept of these clades is supported mainly by phylogenetic and ecological evidence. The inclusion of freshwater representatives of the Plagioporinae sensu Cribb, Reference Cribb, Jones, Bray and Gibson2005 (S. bramae and N. skrjabini) into the clade ‘freshwater Plagioporinae’ (fig. 2) was quite predictable, taking into account the last of the above-mentioned criteria.
The distribution of the remaining plagioporines studied – M. muraenolepidis and P. atomon – was difficult to predict. Macvicaria muraenolepidis appeared in the clade ‘marine Plagioporinae C’, as a sister to M. magellanica (fig. 2). At the same time, most Macvicaria Gibson & Bray, 1982 with known 28S rDNA sequences belonged to the clade Opistholebetinae (Rima et al., Reference Rima2017; Martin et al., Reference Martin, Cutmore and Cribb2017, Reference Martin2018a, Reference Martinb, Reference Martin, Cutmore and Cribbc). Both species under consideration are in different morphological groups of Macvicaria spp. (by Aken'Ova et al., Reference Aken'Ova, Cribb and Bray2008): group D (M. muraenolepidis) and group B (M. magellanica) (Zdzitowiecki, Reference Zdzitowiecki1990b; Aken'Ova et al., Reference Aken'Ova, Cribb and Bray2008; Laskowski et al., Reference Laskowski, Jeżewski and Zdzitowiecki2013). However, both belong to the same zoogeographical group, namely the Southern Ocean fauna (Laskowski et al., Reference Laskowski, Jeżewski and Zdzitowiecki2013).
Our analysis confirms the data provided by Martin et al. (Reference Martin2018a) on the close relationship between the Antarctic and Subantarctic Macvicaria spp. with representatives of the genus Choerodonicola Cribb, 2005. According to Martin et al. (Reference Martin2018a) the genus Choerodonicola is paraphyletic. According to our data, its paraphyly is unclear because of low support of the direct sister relationship of the M. muraenolepidis + M. magellanica group with C. renko. Like Martin et al. (Reference Martin2018a), we do not think there are enough data to put Antarctic and Subantarctic Macvicaria spp. into the genus Choerodonicola today. Firstly, revision of Macvicaria genus is impossible without molecular data for its type species Macvicaria alacris (Looss, 1901). The second reason is the uncertainty regarding the revealed relationships between the above-mentioned Macvicaria spp. and C. renko.
In general, our data indicate a polyphyly not only of the genus Macvicaria, but also three other genera of opecoelids: Allopodocotyle Pritchard, 1966, Podocotyloides Yamaguti, 1934 and Gaevskajatrema Gibson & Bray, 1982 (fig. 2), which is in full accordance with the data in the literature (Andres et al., Reference Andres, Pulis and Overstreet2014; Shedko et al., Reference Shedko, Sokolov and Atopkin2015; Bray et al., Reference Bray2016; Faltýnková et al., Reference Faltýnková, Klimpel, Kuhn and Mehlhor2017; Martin et al., Reference Martin, Cutmore and Cribb2017, Reference Martin, Cutmore and Cribb2018c; Rima et al., Reference Rima2017).
According to Fayton and Andres (Reference Fayton and Andres2016), and Martin et al. (Reference Martin2018b) three сlades of the plagioporine species – ‘marine Plagioporinae B’, ‘marine Plagioporinae C’ and ‘freshwater + deep-sea Plagioporinae’ – require recognition as independent subfamilies. However, we should not forget that there are still no molecular data on indisputable representatives of the Stenakrinae (see Sokolov et al., Reference Sokolov2017), as well as for opecoelinines, which may change the topology of the tree for the Opecoelidae. Thus, the question about the taxonomic status of the three listed clades remains open.
Acknowledgments
The authors wish to thank S. A. Denisova (SPbSU) and the crew of FV Anatoly Torchinov for help with material collection. The work was performed at the Research Park of St. Petersburg State University Centre for Molecular and Cell Technologies.
Financial support
This study was funded by the Russian Science Foundation (grant 17-74-10203).
Conflict of interest
None.
Ethical standards
All applicable international, national and/or institutional guidelines for the care and use of animals were followed by the authors. All necessary permits for sampling and observational field studies were obtained by the authors from the competent authorities.
