Marine Biology Research

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/smar20

Phylogenetic position of the hemiuroid genus Paraccacladium Bray & Gibson, 1977 (: Hemiuroidea) and the status of the subfamily Paraccacladiinae Bray & Gibson, 1977

Sergey G. Sokolov, Dmitry M. Atopkin & Ilya I. Gordeev

To cite this article: Sergey G. Sokolov, Dmitry M. Atopkin & Ilya I. Gordeev (2021): Phylogenetic position of the hemiuroid genus Paraccacladium Bray & Gibson, 1977 (Trematoda: Hemiuroidea) and the status of the subfamily Paraccacladiinae Bray & Gibson, 1977, Marine Biology Research, DOI: 10.1080/17451000.2021.1891252 To link to this article: https://doi.org/10.1080/17451000.2021.1891252

Published online: 10 Mar 2021.

Submit your article to this journal

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=smar20 MARINE BIOLOGY RESEARCH https://doi.org/10.1080/17451000.2021.1891252

ORIGINAL ARTICLE Phylogenetic position of the hemiuroid genus Paraccacladium Bray & Gibson, 1977 (Trematoda: Hemiuroidea) and the status of the subfamily Paraccacladiinae Bray & Gibson, 1977 Sergey G. Sokolov a, Dmitry M. Atopkin b and Ilya I. Gordeev c,d aA.N. Severtsov Institute of Ecology and Evolution, Moscow, Russia; bFederal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch of the RAS, Vladivostok, Russia; cPacific Salmons Department, Russian Federal Research Institute of Fisheries and Oceanography, Moscow, Russia; dDepartmant of Invertebrate Zoology, Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia

ABSTRACT ARTICLE HISTORY In this study we tested the current taxonomic model of the trematode superfamily Received 20 December 2020 Hemiuroidea, according to which the genus Paraccacladium belongs to the family Accepted 5 February 2021 Accacoeliidae. We reconstructed the phylogeny of the hemiuroid Clade A using novel 28S KEYWORDS rRNA gene sequences of Paraccacladium sp., Derogenes varicus, Progonus muelleri and Derogenes varicus; Progonus Lampritrema miescheri as well as data available in GenBank. Based on phylogenetic data, the muelleri;·Lampritrema genus Paraccacladium should be assigned to a separate family, the Paraccacladiidae Bray & miescheri; Paraccacladiidae; Gibson, 1977 stat. nov. The morphological differences between the Paraccacladiidae and the Accacoeliidae; Accacoeliidae are the absence of a uroproct, anteriorly directed diverticula of intestinal caeca, and the position of Mehlis’ gland, posterior or postero-lateral to the ovary. Phylogenetic analysis indicates a sister relationship between the Derogenidae and the Sclerodistomidae. It also shows that the Paraccacladiidae, on the one hand, and a group of families, the Accacoeliidae, Hirudinellidae and Syncoeliidae, on the other, share the most recent common ancestor. Our data support the hypothesis that the genus Lampritrema is affiliated to the Hirudinellidae. Genetic divergence between North Atlantic and North Pacific isolates of D. varicus was also determined.

Introduction on a short fragment of the 18S rRNA gene (Pankov Paraccacladium Bray & Gibson, 1977 is a small genus of et al. 2006) did not reveal any direct phylogenetic hemiuroid trematodes. It contains only two species, relationships between the Paraccacladiinae and the Paraccacladium jamiesoni Bray & Gibson, 1977 (type nominotypical subfamily of the Accacoeliidae. At the species) and Paraccacladium leontjevae (Korotaeva, same time, the Accacoeliinae together with the 1976) (see Bray and Gibson 1977). Paraccacladium families Derogenidae, Didymozoidae, Hirudinellidae, jamiesoni is typical of the North and Central Atlantic Sclerodistomidae and Syncoeliidae were found to (Bray and Gibson 1977; Campbell et al. 1980; Gaevs- belong to a monophyletic group (Clade A) in 28S kaya and Aleshkina 1983; Scott 1987; Gaevskaya rRNA gene-based phylogeny of the Hemiuroidea 2002) but was also recorded in the northwest-central (Sokolov et al. 2019). region of the North Pacific (Machida 1985). Paraccacla- During the course of a parasitological survey of dium leontjevae was found in the South Pacific (Koro- fishes and amphipods caught in the North-west taeva 1976) and South Atlantic (Aleshkina and Pacific and the White Sea, we found Paraccacladium Gaevskaya 1985). sp., as well as the derogenids Derogenes varicus In the current taxonomic model of the Hemiuroidea, (Müller, 1784) (North Pacific isolate) and Progonus Paraccacladium is a member of the monotypic subfam- muelleri (Levinsen, 1881), and a hirudinellid Lampri- ily Paraccacladiinae Bray & Gibson, 1977 of the family trema miescheri (Zschokke, 1890). These trematodes Accacoeliidae Odhner, 1911 (see Gibson 2002). have never been studied with the use of 28S rDNA However, the results of phylogenetic analysis based sequence data. The goal of the present study was to

CONTACT ILYA I. GORDEEV [email protected] Pacific Salmons Department, Russian Federal Research Institute of Fisheries and Oceanography, V. Krasnoselskaya Str. 17, Moscow, 107140, Russia Departmant of Invertebrate Zoology, Faculty of Biology, Lomonosov Moscow State University, Leninskie Gory 1/12, Moscow, 119234, Russia Supplemental data for this article can be accessed https://doi.org/10.1080/17451000.2021.1891252 © 2021 Informa UK Limited, trading as Taylor & Francis Group

Published online 10 Mar 2021 2 S. G. SOKOLOV ET AL. reveal the taxonomic status of the subfamily Paracca- descriptions are given in micrometres, unless stated cladiinae based on the 28S rRNA gene-based phyloge- otherwise. Drawings of Derogenes varicus, Lampritrema netic analysis of the trematodes from our collections miescheri and Progonus muelleri are in the Supplemen- and the GenBank (NCBI) dataset. tary Materials (Figures S1–S3). The identification of these species was undertaken following Margolis (1962) and Gibson (1996). Slides with trematodes Materials and methods were deposited in the Museum of Helminthological Collections at the Center of Parasitology of the A.N. Sample collection and morphological Severtsov Institute of Ecology and Evolution (IPEE observation RAS) in Moscow, Russia. Specimens for molecular The trematodes were collected during a parasitological analysis were fixed in 96% ethanol and stored at examination of six species of North Pacific fishes: Apto- −18°C. cyclus ventricosus (Pallas, 1769) (Actinopterygii: Cyclop- teridae), total length 2.9–25 cm, weight 12–1385 g; DNA extraction, amplification and sequencing, Icichthys lockingtoni Jordan & Gilbert, 1880 (Actinopter- and phylogenetic analysis ygii: Centrolophidae), 42 cm, 512 g; Leuroglossus schmidti Rass, 1955 (Actinopterygii: Bathylagidae), Total DNA was extracted from the worms fixed in 96% 11.1–17.2 cm, 8–12 g; Brama japonica Hilgendorf, ethanol using the ‘hot shot’ technique (Truett 2006). 1878 (Actinopterygii: Bramidae), 37.7–47.0 cm, 1086– The polymerase chain reaction (PCR) was used to 1327 g; Eumicrotremus fedorovi Mandrytsa, 1991 (Acti- amplify V4 region of 18S rRNA gene using the nopterygii: Cyclopteridae), 6.7–8.9 сm, 12.4–45.2 g; and primers 18S-8 (5’-GCA GCC GCG GTA ACT CCA GC-3’) Antimora microlepis Bean, 1890 (Actinopterygii: and 18S-A27 (5’-CCA TAC AAA TGC CCC CGT CTG-3’) Moridae), 11.5–45.2 cm, 4–1350 g. as previously described (Littlewood and Olson 2001). Aptocyclus ventricosus, I. lockingtoni and L. schmidti The initial PCR was performed in a total volume of 25 were caught from the research vessel Professor Kaga- µl and contained 0.25 mM of each primer, ∼10 ng of novsky between 31 May 2018 and 7 July 2018 in the total DNA in water and 12.5 µl of 2× Go Taq Green open waters of the North-west Pacific (40–50°N, 147– Master mix (Promega). Amplification of a 400 base 167°E) (Gordeev et al. 2018; Gordeev and Sokolov pair (bp) fragment of 18S rDNA was performed in a 2020b). Brama japonica was caught from the research GeneAmp 9700 (Applied Biosystems, USA) with a 5 vessel Professor Kaganovsky on 27 September 2019 in min denaturation at 96°C, 35 cycles of 1 min at 96°C, the Bering Sea (53°38′05′′N, 169°23′05′′E) (Gordeev 20 s at 58°C and 1 min at 72°C and a 2 min extension et al. 2019). Antimora microlepis was caught in two at 72°C. Negative and positive controls with the use localities: near Japan (41–42°N, 143–144°E) between of both primers were included. 7 and 10 June 2016 at a depth from 560–961 m and The 28S rRNA gene was amplified with the primers from RV Professor Levanidov on 2 September 2018 DIG12 (5’-AAG CAT ATC ACT AAG CGG-3’) and 1500R near the Kuril Islands (49°34′N, 156°19′E) at a depth (5’-GCT ATC CTG AGG GAA ACT TCG-3’) as previously of 550 m. Eumicrotremus fedorovi was caught near described (Tkach et al. 2003). The master mix for the Simushir Island (47°13′04′′N, 152°27′06′′E; 46°44′08′′N, PCR reaction was identical to that described above 151°52′05′′E) on 23–24 March 2017 at a depth of 230 for 18S rDNA. Amplification of a 1200 bp fragment m (Gordeev and Sokolov 2020a). Specimens of An. of 28S rRNA gene was carried out in a GeneAmp microlepis and E. fedorovi were frozen, and then 9700 (Applied Biosystems, USA) with a 3 min dena- thawed and dissected in the laboratory, while the turation at 94°C, 40 cycles of 30 s at 94°C, 30 s at other specimens were dissected directly after capture. 55°C and 2 min at 72°C and a 7 min extension at Trematodes were also isolated from the amphipod 72°C. Negative and positive controls with the use of Caprella septentrionalis Krøyer, 1838 (Amphipoda: both primers were included. A ribosomal ITS1-5.8S- Caprellidae) collected near the White Sea Biological ITS2 fragment was amplified with primers BD1 (5’- Station of Lomonosov Moscow State University GTC GTA ACA AGG TTT CCG TA-3’) and BD2 (5’-TAT (Great Salma Straight, White Sea, 66°33′01′′N, 33° GCT TAA ATT CAG CGG GT-3’) (Luton et al. 1992) 09′10′′E) on 19 July 2019. with an annealing temperature of 54°C. Negative For morphological study the worms were fixed in and positive controls with the use of both primers 70% ethanol, stained with acetocarmine, cleared in were included. dimethyl phthalate and mounted in Canadian PCR products were directly sequenced using the ABI balsam. All of the measurements in the morphological Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied MARINE BIOLOGY RESEARCH 3

Biosystems, USA), as recommended by the manufac- Site of infection turer, with the internal sequencing primers described Intestine. by Littlewood and Olson (2001) for the 18S rRNA gene, by Tkach et al. (2003) for 28S rRNA gene and by Luton et al. (1992) for the ITS1 DNA fragment. PCR Locality products were analysed using Genetic Analyzer 3500 45°09′03′′–45°56′03′′ N, 152°18′07′′ E–153°20′05′′ E(Ap. (Applied Biosystems, USA) at the Federal Scientific ventricosus); 48°13′02′′ N, 162°26′00′′ E(Ap. ventricosus); Center of Biodiversity FEB RAS. Sequences were sub- 43°26′04′′ N, 155°56′02′′ E(I. lockingtoni); 50°06′04′′ N, mitted to GenBank of the NCBI database. Accession 158°50′09′′ E(L. schmidti); 49°34′ N, 156°19′ E and 41– numbers are given in Table 1. 42° N, 143–144° E (An. microlepis). The rDNA sequences were assembled with SeqS- cape v. 2.6 software. Alignments and estimation of the number of variable sites and the estimation of p- Prevalence distances were performed using MEGA 7.0 (Kumar ′ ′′– et al. 2016). Phylogenetic analyses of the 28S rRNA Two of two Ap. ventricosus specimens (45°09 03 45° 56′03′′ N, 152°18′07′′ E–153°20′05′′ E); one of two Ap. gene sequences were performed using the Maximum ′ ′′ ′ ′′ likelihood (ML) and the Bayesian (BI) algorithms with ventricosus specimens (48°13 02 N, 162°26 00 E); the PhyML 3.1 (Guindon and Gascuel 2003) and one of one I. lockingtoni specimen; 2 of 13 L. schmidti – MrBayes v. 3.1.2 software (Huelsenbeck et al. 2001), specimens; 10 of 25 An. microlepis specimens (41 42° – respectively. The best nucleotide substitution models N, 143 144° E) and 1 of 20 An. microlepis specimens ’ ’ GTR + I+G and TVM + G were estimated with jModelt- (49°34 N, 156°19 E). est v. 2.1.5 software (Darriba et al. 2012) for Maximum likelihood and Bayesian algorithms, respect- Intensity of infection ively. Bayesian analysis was performed using 10,000,000 generations, with two independent runs. One worm in Ap. ventricosus; one worm in I. lockingtoni; – – Summary parameters and the phylogenetic tree were 1 3 worms in L. schmidti;1138 worms in An. calculated with a burn-in of 2,500,000 generations. microlepis. The significance of the phylogenetic relationships was estimated using posterior probabilities (Huelsen- Deposited material beck et al. 2001) for both Maximum likelihood and Bayesian algorithms. The phylogenetic relationships Two paragenophores ex An. microlepis, IPEE RAS were inferred using our samples and trematode 14290. species of the hemiuroid Clade A (after Sokolov et al. 2019) obtained from the NCBI GenBank database Representative DNA sequences (Table 2). The six partial 28S rRNA gene sequences, six V4 region of the 18S rRNA gene sequences and four sequences of Results ITS1-5.8S-ITS2 fragment were deposited in GenBank (NCBI) (Table 1). Systematics

Superfamily Hemiuroidea Looss, 1899 Description Family Paraccacladiidae Bray & Gibson, 1977 stat. nov. [Based on four paragenophores ex An. microlepis: one Genus Paraccacladium Bray & Gibson, 1977 fully gravid and three subgravid specimens]. – Paraccacladium sp., fully gravid, subgravid and imma- Body elongate, smooth; length 2.04 3.39 mm; – ture specimens maximum width 0.56 0.70 mm at level of ventral – (Figures 1A–C; 2A, B) sucker. Oral sucker rounded quadriangular, 207 281 × 220–244, subterminal. Ventral sucker rounded or ovoid, 390–476 × 342–366, protuberant, with longi- tudinal aperture. Oral sucker to ventral sucker width Hosts ratio 1:1.50–1.56. Forebody 26.0–31.8% of body Aptocyclus ventricosus, Icichthys lockingtoni, Leuroglos- length. Prepharynx absent. Pharynx pyriform, 110– sus schmidti and Antimora microlepis. 122 × 85, protruding into base of oral sucker. 4 S. G. SOKOLOV ET AL.

Table 1. List of examined hemiuroid species; n – number of specimens. NCBI accession number ITS1-5.8S-ITS2 rDNA Species n Host Location 28S rDNA locus V4 of 18 rDNA locus locus Paraccacladiidae Paraccacladium sp. 1 Aptocyclus 45°09′03′′ N, 152°18′07′′ MW507465 MW528738 MW528749 ventricosus E 1 Aptocyclus 48°13′02′′ N, 162°26′00′′ MW507466 MW528739 MW528750 ventricosus E 1 Leuroglossus schmidti 50°06′04′′ N, 158°50′09′′ MW507467 MW528740 MW528751 E 1 Icichthys lockingtoni 43°26′04′′ N, 155°56′02′′ MW507468 MW528741 MW528752 E 2 Antimora microlepis 42°15′58′′ N & 143° MW507463; MW528834; – 58′08′′ E MW507464 MW528835 Derogenidae Derogenes varicus 2 Eumicrotremus 47°13′04′′ N, 152°27′06′′ MW504598 –– fedorovi E 46°44′08′′N,151°52′05′′ E MW504599 –– Progonus muelleri 2 Eumicrotremus 46°44′08′′ N, 151°52′05′′ MW507469; –– fedorovi E MW507470 1 Caprella 66°33′01′′ N, 33°09′10′′ E MW507471 –– septentrionalis Hirudinellidae Lampritrema 1 Brama japonica 53°38′05′′ N, 169°23′05′′ MW507472 –– miescheri E

Oesophagus rounded, 85–61 × 61, thick-walled. Caeca Molecular data and phylogenetic analysis simple, sinuous, terminating blindly close to posterior We obtained four ribosomal ITS1-5.8S-ITS2 fragments, extremity of body. each 1063 bp in length, for Paraccacladium sp. from Testes two, oval, tandem or slightly oblique, contig- three host species, Aptocyclus ventricosus, Leuroglossus uous; both in anterior half of hindbody, although pos- schmidti and Icichthys lockingtoni. All the four terior testis may occasionally extend slightly into sequences were identical. posterior half; anterior testis 207–305 × 244–281; pos- The sequences of the V4 region of the 18S rRNA terior testis 195–281 × 244–293. Seminal vesicle gene (396 bp) and 28S rRNA gene (1011 bp) from six tubular, convoluted, dorsal to ventral sucker. Pars pros- specimens of Paraccacladium sp. collected from tatica tubular, surrounded by dense layer of prostatic various hosts and localities were used in the analysis gland cells. Ejaculatory duct unites with metraterm (see Table 1). Sequences of the V4 region in our speci- within sinus-sac and forms hermaphroditic duct mens were identical to each other, and had a high which extends into sinus-organ. Sinus-sac surrounds degree of similarity with the 291 bp sequence of base of genital atrium. Sinus-organ permanent, cylind- P. jamiesoni from GenBank (AF029820). The only differ- rical, within relatively large genital atrium. Genital pore ence between the sequences of these trematodes was at level of oesophagus, sometimes at posterior margin T/C transition in 113 position. of pharynx. The BI and ML analyses of the Clade A of 28S rRNA Ovary oval, 171–281 × 171–183, overlaps posterior gene partial sequences indicate that Paraccacladium testis dorsally by 21–77% of its length. Oviduct with sp. is a member of the group that also includes Accacoe- proximal ovicapt. Laurer’s canal inflated proximally, lium contortum (Rudolphi, 1819) (Accacoeliinae, Acca- opens dorso-medially at level of ovary; canalicular coeliidae), Copiatestes filiferus (Leukart in Sars, 1885) seminal receptacle absent. Mehlis’ gland postero- (Syncoeliidae), Lampritrema miescheri and Hirudinella lateral or strictly posterior to ovary, contiguous. spp. (Hirudinellidae). The species under study occupies Uterus descends to level between posterior fourth a basal position in this group and does not form any and posterior fifth of hindbody (in fully gravid speci- direct phylogenetic relationships with A. contortum, men). Eggs numerous, 38 × 22 (in fully gravid speci- the type species of type genus of the family Accacoelii- men). Vitellarium in form of two lateral branching dae (Figure 3). Two of the six 1011 bp 28S rRNA gene tubular systems, extending from testicular level to sequences of Paraccacladium sp. have a single variable about middle, occasionally second quarter of, post- site (no. 588) containing the T/C transition. ovarian region. Common vitelline duct ventral to occupies a sister position oviduct. Excretory vesicle Y-shaped; stem very short; to the well-supported L. miescheri + Hirudinella spp. arms unite dorsally to pharynx. MARINE BIOLOGY RESEARCH 5

Table 2. List of previously published sequences used in the phylogenetic analysis. 28S rRNA gene NCBI Taxa accession. number Reference Accacoeliidae Accacoelium contortum AY222190 Olson et al. (2003) (Rudolphi, 1819) Derogenidae Halipeginae Allogenarchopsis MH628313 Sokolov et al. (2019) problematica (Faust, 1924) Halipegus sp. MK648278 Pérez-Ponce de Leon and Hernández- Mena (2019) Genarchella sp. 1 MK648276 Pérez-Ponce de Leon and Hernández- Mena (2019) Genarchella sp. 2 MK648277 Pérez-Ponce de Leon and Hernández- Mena (2019) Genarchopsis chubuensis MH628311 Sokolov et al. (2019) Shimazu, 2015 Genarchopsis goppo Ozaki, KX344073 [as Chaudhary et al. 1925 Genarchopsis sp.] (2016) Thometrema lotzi Stephen, KC985236 Calhoun et al. (2013) Overstreet & Font, 2002 Derogenidae gen. sp. KX759627 Collicutt et al. (2017) Derogeninae Derogenes varicus (Müller, AY222189 Olson et al. (2003) 1784) Didymozoidae Didymozoon scombri AY222195 Olson et al. (2003) Figure 1. Subgravid Paraccacladium sp. ex Antimora microle- Taschenberg, 1879 Didymocystis scomberomori KU341979 Schrandt et al. (2016) pis. (A) Paragenophore, lateral view; (B) Terminal genitalia, (G. et W. MacCallum, 1916) lateral view; (C) Oral sucker and pharynx, lateral view. Abbrevi- Didymocystis sp. KU341980 Schrandt et al. (2016) ations: ga, genital atrium; pp, pars prostatica; sc, sinus-sac; so, Philopinna higai Yamaguti, MH628312 Sokolov et al. (2019) sinus-organ; sv, seminal vesicle; ut, uterus. Scale bars: A, 1 mm, 1936 Wedlia sp. LC533952 Mekata,T. and Kawato, B, 0.2 mm, C, 0.25 mm. Y. (Direct Submission) Didymozoidae gen. sp. 1 AY222193 Olson et al. (2003) group was represented by Hirudinella ahi Yamaguti, Didymozoidae gen. sp. 2 AY222192 Olson et al. (2003) 1970, Hirudinella sp. A of Calhoun et al. (2013), Hiru- Didymozoidae gen. sp. 3 AY222194 Olson et al. (2003) dinella ventricosa (Pallas, 1774) of Calhoun et al. Didymozoidae gen.sp. MK558797 Vidal-Martínez et al. (2019) (2013) and H. ventricosa of Pérez-Ponce de Leon Hirudinellidae and Hernández-Mena (2019). The latter two samples Hirudinella ahi Yamaguti, KC985238 Calhoun et al. (2013) 1970 are clearly not conspecific. At the same time, Hirudi- Hirudinella ventricosa (Pallas, KC985232 Calhoun et al. (2013) nella sp. B of Calhoun et al. (2013) does not have a 1774) MK648294 Pérez-Ponce de Leon direct phylogenetic relationship with other Hirudinella and Hernández- spp. and appears to be a sister taxon to the Mena (2019) Hirudinella sp. A KC985230 Calhoun et al. (2013) A. contortum +(L. miescheri + other Hirudinella spp.) Hirudinella sp. B KC985233 Calhoun et al. (2013) clade (Figure 3). Sclerodistomidae The clade that unites A. contortum, C. filiferus, Prosogonotrema bilabiatum AY222191 Olson et al. (2003) Vigueras, 1940 L. miescheri, Hirudinella spp. and Paraccacladium KU527431 Claxton et al. (2017) sp. is a poorly supported sister clade to the well- Syncoeliidae Copiatestes filiferus (Leuckart AY222188 Olson et al. (2003) supported group containing the derogenine, hali- in Sars, 1885) pegine and sclerodistomid hemiuroids. In turn, Outgroup Gonocercidae the derogenines and halipegines appear to be Gonocerca oshoro Shimazu, KY197013 Sokolov et al. (2018) sister groups, with a moderate support in the BI 1970 analysis and a low support in the ML analysis (Figure 3). Nucleotide sequences of 28S rRNA gene, obtained group, but this sister relationship has no reliable from specimens of Progonus muelleri from the White support in BI and ML analyses. The Hirudinella spp. Sea and the North Pacific, were identical. At the same subgroup within the L. miescheri + Hirudinella spp. time, Derogenes varicus isolate from the North Pacific 6 S. G. SOKOLOV ET AL.

Figure 2. Proximal part of the female system of Paraccacladium sp. (A) Ovary and Mehlis’ gland adjacent to its postero-sinistral edge, ventral view; (B) ovarian complex, ventral view. Abbreviations: cvd, common vitelline duct, elc, extended proximal part of Laurer’s canal; lc, Laurer’s canal; mg, Mehlis’ gland; oc, oocapt, ot, ootype; ov, ovary; ovd, oviduct; ut, uterus. Scale bars: A, 0.4 mm; B, 0.05 mm. differed from that from the North Atlantic by 11 substi- of subgravid specimens was 2.20–3.39 mm), we tutions (р-distance = 0.97 ± 0.3%). believe that the recorded egg sizes are not modal for our isolate. Unlike P. jamiesoni from the type host (Coryphae- Discussion noides rupestris Gunnerus, 1765, Macrouridae), our tre- The trematode specimens described in this paper matodes lack the dome-shaped papillae on the outer clearly belong to the genus Paraccacladium based on surface of the ventral sucker (Bray and Gibson 1977). the morphology of the oral sucker, pharynx, intestinal Perhaps this difference is due to the exfoliation of caeca, vitellarium, sinus-organ and terminal portion of the tegument during the freezing and thawing of the the male terminal genitalia as well as the position of samples (see Materials and methods). Machida (1985) Mehlis’ gland (see Bray and Gibson 1977; Machida also found P. jamiesoni in Antimora microlepis and 1985; Gibson 2002). Aptocyclus ventricosus from the North Pacific, but The two species of Paraccacladium, P. jamiesoni and only the specimens from the latter of these hosts P. leontjevae,differ morphologically from each other in were gravid. The specimens described by Machida the size of the eggs (Bray and Gibson 1977; Korotaeva (1985) also had no papillae on the outer surface of 1976) and possibly also the presence/absence of an the ventral sucker. oesophagus, although the absence of the oesophagus The analysis of the V4 region of 18S rRNA gene in P. leontjevae, mentioned in the original description showed that the trematodes in our study are phylo- (Korotaeva 1976), was questioned by Bray and genetically close to the P. jamiesoni and are probably Gibson (1977). With regard to the length of the eggs, conspecific with it. Nevertheless, taking into account our specimens are similar to P. leontjevae (36 × the morphological features of our specimens men- 16 μm), but in the width of the eggs they are closer tioned above, we identify them as Paraccacladium sp. to P. jamiesoni (44–59 × 22–29μm) (Bray and Gibson and provide molecular data on the ribosomal ITS1- 1977; Machida 1985; Gaevskaya 2002). Gravid speci- 5.8S-ITS2 sequences, which can be used to assess mens of P. jamiesoni described in the literature are their taxonomic relations with P. jamiesoni in the ≥3.08 mm long (Bray and Gibson 1977; Machida future. 1985; Gaevskaya 2002). In our sample, egg sizes were Paraccacladium is the type and the only genus of measured in a single fully gravid specimen with a the subfamily Paraccacladiinae (Bray and Gibson body length of only 2.04 mm. Taking into account 1977; Gibson 2002). The most striking morphological that reaching a fully gravid stage at this body length differences between the Paraccacladiinae and the is an exception rather than the rule for the studied nominotypical subfamily of the Accacoeliidae are isolate of Paraccacladium (for comparison, the length the absence or presence of a uroproct, anteriorly MARINE BIOLOGY RESEARCH 7

Figure 3. Phylogenetic relationships of the hemiuroids of Clade A, reconstructed by Maximum likelihood and Bayesian inference analysis of 28S rRNA gene sequences. References for the data retrieved from GenBank are listed in Table 2. Posterior probabilities (ML/BI) of less than 0.9 are not indicated. Newly obtained sequences are marked in bold. directed diverticula of intestinal caeca and the pos- study). Our phylogenetic data combined with the ition of Mehlis’ gland (posterior or postero-lateral to results of Pankov et al. (2006), together the above- the ovary vs pre-ovarian) (Bray and Gibson 1977; mentioned morphological and ecological differences Machida 1985; Gibson 2002). Moreover, the between the paraccacladiine and accacoeliine trema- members of the Paraccacladiinae differ from todes, indicate that the Paraccacladiinae should be members of most genera of the Accacoeliinae, such considered as an independent family, the Paraccacla- as species of Accacoelium Monticelli, 1893, Accada- diidae Bray & Gibson, 1977 stat. nov. The diagnosis of dium Odhner, 1928, Accacladocoelium Odhner, 1928, the family Paraccacladiidae coincides with that of the Rhynchopharynx Odhner, 1928, Odhnerium Yamaguti, subfamily Paraccacladiinae (see Gibson 2002). 1934 and Orophocotyle Looss, 1902, in ecological fea- Our data detail the topology of the hemiuroid Clade tures. Representatives of these six genera are known А of Sokolov et al. (2019) and indicate the existence of only from ocean sunfishes (Molidae) (Bray and Gibson the most recent common ancestor of the Paraccacladii- 1977; Gibson 2002), whereas paraccacladiines are dae and the hemiuroid group that unites the families known from a wide range of fishes, such as macrour- Accacoeliidae, Hirudinellidae and Syncoeliidae. Our ids, morids, alepocephalids, cyclopterids and others data also support the validity of combining the dero- (Korotaeva 1976; Bray and Gibson 1977; Gaevskaya genines and halipegines within the same family, the and Aleshkina 1983; Aleshkina and Gaevskaya 1985; Derogenidae (e.g. Gibson 2002), and point to the exist- Machida 1985; Scott 1987; Gaevskaya 2002; Present ence of the most recent common ancestor of the 8 S. G. SOKOLOV ET AL. derogenids and sclerodistomids. A sister relationship Disclosure statement between the Derogenidae and Sclerodistomidae and No potential conflict of interest was reported by the authors. the monophyletic origin of the group of families con- taining the Paraccacladiidae, Accacoeliidae, Hirudinel- lidae and Syncoeliidae are not supported by any of Funding the morphology-based taxonomic hypotheses for This work is a part of the state-supported studies in the A.N. these hemiuroid trematodes (Gibson and Bray 1979; Severtsov Institute of Ecology and Evolution of RAS (project Brooks et al. 1985). We also fail to see any morphologi- no. 0109–2018–0075) and the Federal Scientific Center of cal synapomorphies for each of these two evolutionary the East Asia Terrestrial Biodiversity of FEB RAS (project no. lineages. 0267-2019-0018). Our data support the taxonomic hypothesis Gibson and Bray (1977) that the genus Lampritrema ORCID Yamaguti, 1940 belongs to the family Hirudinellidae. Sergey G. Sokolov http://orcid.org/0000-0002-4822-966X At the same time, the affiliation of Hirudinella sp. B Dmitry M. Atopkin http://orcid.org/0000-0001-8417-3424 of Calhoun et al. (2013) with this family and the Ilya I. Gordeev http://orcid.org/0000-0002-6650-9120 genus Hirudinella de Blainville, 1828 is doubtful. 28S rRNA gene-based molecular differences of Hirudinella sp. B (KC985233, 995 bp) from other sequenced Hiru- References dinella spp. (991 bp) are as follows: five-nucleotide Aleshkina LD, Gaevskaya AV. 1985. Trematodes of fish from deletion at positions 539–543, single deletions at pos- the Atlantic coast of Africa. Nauchnye Doklady Vysshei itions 522 and 551, fourth-nucleotide insertion at pos- Shkoly. Biologicheskie Nauki. 3:35–40 (in Russian). itions 536–538 and the presence of hypervariable Atopkin DM, Sokolov SG, Vainutis KS, Voropaeva EL, Shedko MB, Choudhury A. 2020. Amended diagnosis, validity and parts along the sequence, with p-distance values relationships of the genus Acrolichanus Ward, 1917 ranging from 7.5 ± 0.7% to 7.7 ± 0.7%. The lack of (: Allocreadiidae) based on the 28S rRNA gene, detailed morphological data for this parasite pre- and observation on its lineage diversity. Systematic cludes a more substantial discussion of its systematic Parasitology. 97:143–156. doi:10.1007/s11230-020- position. 09901-z. The divergence of two isolates of Derogenes Bray RA, Gibson DI. 1977. The Accacoeliidae (Digenea) of fishes from the North-East Atlantic. Bulletin of the British varicus, revealed in this study based on molecular Museum (Natural History), (Zoology Series). 31:53–99. data, is remarkable. The genetic p-distance value is Brooks DR, O’Grady RT, Glen DR. 1985. Phylogenetic analysis close to the boundary values of intra- and inter- of the Digenea (Platyhelminthes: Cercomeria) with com- specific divergence level for trematodes, estimated ments on their adaptive radiation. Canadian Journal of – by means of 28S rRNA gene (Marzoug et al. 2014; Zoology. 63:411 443. doi:10.1139/z85-062. Calhoun DM, Curran SS, Pulis EE, Provaznik JM, Franks JS. Sokolov et al. 2018; Atopkin et al. 2020). It cannot 2013. Hirudinella ventricosa (Pallas, 1774) Baird, 1853 rep- be ruled out that this taxon is in fact a complex of resents a species complex based on ribosomal DNA. cryptic species. Systematic Parasitology. 86:197–208. doi:10.1007/s11230- Thus, the family Accacoeliidae sensu Bray & Gibson 013-9439-2. (1977) is polyphyletic. This problem can be solved by Campbell RA, Haedrich RL, Munroe TA. 1980. Parasitism and fi removing the Paraccacladiinae from the Accacoeliidae ecological relationships among deep-sea benthic shes. Marine Biology. 57:301–313. doi:10.1007/BF00387573. and elevating this subfamily to family status. Chaudhary A, Mukut S, Singh HS. 2016. Molecular character- ization of three species belongs to the Allocreadioidea, Hemiuroidea and Plagiorchioidea (Platyhelminthes: Acknowledgements Trematoda) infecting freshwater fishes in India. Helminthologia. 53:378–384. doi:10.1515/helmin-2016- The authors are grateful to the crew of the RV Professor Kaga- 0039. novsky (VNIRO) and to Dr Valeriy Shevlyakov (Pacific Ocean Claxton AT, Fuehring AD, Andres MJ, Moncrief TD, Curran SS. Branch of the Russian Federal Research Institute of Fisheries 2017. Parasites of the Vermilion Snapper, Rhomboplites and Oceanography) for help with the collection and trans- aurorubens (Cuvier, 1829), from the western Atlantic portation of the specimens. Ocean. Comparative Parasitology. 84:1–14. doi:10.1654/ 1525-2647-84.1.1. Collicutt NB, Stacy NI, Walden HS, Childress A, Dill J, Anderson Data availability statement M, Wellehan JFX. 2017. Infection with a novel derogenid trematode in a flap-necked chameleon (Chamaeleo The data that support the findings of this study are openly dilepis). Veterinary Clinical Pathology. 46:629–634. doi:10. available. 1111/vcp.12537. MARINE BIOLOGY RESEARCH 9

Darriba D, Taboada GL, Doallo R, Posada D. 2012. Littlewood DTJ, Olson PD. 2001. Small subunit rDNA and the Jmodeltest2: more models, new heuristics and parallel platyhelminthes: signal, noise, conflict and compromise. computing. Nature Methods. 9:772. doi:10.1038/nmeth. In: Littlewood DTJ, Bray RA, editors. Interrelationships of 2109. platyhelminthes. London: Taylor & Francis; p. 262–278. Gaevskaya AV. 2002. New data on trematodes of the families Luton K, Walker D, Blair D. 1992. Comparisons of ribosomal Opecoelidae and Accacoelidae from fishes in Atlantic internal transcribed spacer from two congeric species of Ocean and its seas. Parazitologiya. 36:219–223 (in Russian). flukes (Platyhelminthes: Trematoda: Digenea). Molecular Gaevskaya AV, Aleshkina LD. 1983. New data on the trema- Biochemical Parasitology. 56:323–328. doi:10.1016/0166- todes from the fishes of Atlantic coast of Africa. 6851(92)90181-I. Parazitologiya. 17:12–17 (in Russian). Machida M. 1985. Helminth parasites of cyclopterid fish, Gibson DI. 1996. Trematoda. In: Margolis L, Kabata Z, editors. Aptocyclus ventricosus, caught off northern Japan. Guide to the parasites of fishes of Canada, part IV. Ottawa: Bulletin of the National Science Museum, Series A NRC Research Press; p. 1–373. (Zoology). 11:123–128. Gibson DI. 2002. Family Accacoeliidae Odhner, 1911. In: Margolis L. 1962. Lampritrema nipponicum Yamaguti Gibson DI, Jones A, Bray RA, editors. Keys to the (Trematoda) from new hosts in the north Pacific Ocean, Trematoda, volume 1. London: CAB International; the relationship of Distomum miescheri Zschokke, and p. 341–347. the status of the family Lampritrematidae. Canadian Gibson DI, Bray RA. 1977. The Azygiidae, Hirudinellidae, Journal of Zoology. 40:941–950. doi:10.1139/z62-083. Ptychogonimidae, Sclerodistomidae and Syncoeliidae Marzoug D, Rima M, Boutiba Z, Georgieva S, Kostadinova A, (Digenea) of fishes from the north-east Atlantic. Bulletin Pérez-del-Olmo A. 2014. A new species of Saturnius of the British Museum Natural History, Zoology, 32:167– Manter, 1969 (Digenea: ) from Mediterranian 245. mullet (Teleostei: Mugilidae). Systematic Parasitology. Gibson DI, Bray RA. 1979. The Hemiuroidea: terminology. sys- 87:127–134. doi:10.1007/s11230-013-9468-x. tematics and evolution. Bulletin of the British Museum Olson PD, Cribb TH, Tkach VV, Bray RA, Littlewood DTJ. 2003. (Natural History), (Zoology Series). 36:35–146. Phylogeny and classification of the Digenea Gordeev II, Shevlyakov VA, Kurnosov DS, Ponomarev SS, (Platyhelminthes: Trematoda). International Journal for Kozhevnikov AV, Chistyakova TA, Bezverkhnyaya AO, Parasitology. 33:733–755. doi:10.1016/s0020-7519 Zhiltsov AE, Svidersky VA, Sheybak AY. 2019. Trawl (03)00049-3. survey of Pacific salmon on the R/V “Professor Pankov P, Webster BL, Blasco-Costa I, Gibson DI, Littlewood Kaganovsky” in the Bering Sea and Okhotsk Sea DTJ, Balbuena JA, Kostadinova A. 2006. Robinia aurata (September-October 2019). VNIRO Proceedings. 178:200– n.g., n. sp. (Digenea: Hemiuridae) from the mugilid Liza 205 (in Russian). doi:10.36038/2307-3497-2019-178-200- aurata with a molecular confirmation of its position 205. within the Hemiuroidea. Parasitology. 133:217–227. Gordeev II, Sokolov SG. 2020a. Helminths and the feeding doi:10.1017/S0031182006000126. habits of the spiny lumpsucker Eumicrotremus fedorovi Pérez-Ponce de Leon G, Hernández-Mena DI. 2019. Testing Mandrytsa, 1991 (Pisces: Cyclopteridae) in the Simushir the higher-level phylogenetic classification of Digenea Island area (Pacific Ocean). Parasitology International. (Platyhelminthes, Trematoda) based on nuclear rDNA 76:102075. doi:10.1016/j.parint.2020.102075. sequences before entering the age of the ‘next-gener- Gordeev II, Sokolov SG. 2020b. Macroparasites of epipelagic ation’ Tree of Life. Journal of Helminthology. 93:260–276. and eurybathic fishes in the north-western Pacific Ocean. doi:10.1017/S0022149X19000191. Invertebrate Zoology. 17:118–132. doi:10.15298/ Schrandt MN, Andres MJ, Powers SP, Overstreet RM. 2016. invertzool.17.2.02. Novel infection site and ecology of cryptic Didymocystis Gordeev II, Starovoytov AN, Ponomarev SS, Shevlyakov AV, sp. (Trematoda) in the fish Scomberomorus maculates. Milovankin PA. 2018. Trawl survey of Pacific salmon on Journal of Parasitology. 102:297–305. doi:10.1645/15-772. the R/V “Professor Kaganovsky” in the northwestern part Scott JS. 1987. Helminth parasites of the alimentary tract of of the Pacific Ocean (May-July 2018). VNIRO Proceedings. the hakes (Merluccius, Urophycis, Phycis: Teleostei) of the 171:208–221 (In Russian). Scotian Shelf. Canadian Journal of Zoology. 65:304–311. Guindon S, Gascuel O. 2003. A simple, fast, and accurate doi:10.1139/z87-047. algorithm to estimate large phylogenies by maximum like- Sokolov SG, Atopkin DM, Gordeev II, Shedko MB. 2018. lihood. Systematic Biology. 52:696–704. doi:10.1080/ Phylogenetic position of the genus Gonocerca Manter, 10635150390235520. 1925 (Trematoda, Hemiuroidea), based on partial Huelsenbeck JP, Ronquist F, Nielsen R, Bollback JP. 2001. sequences of 28S rRNA gene and a reconsideration of Bayesian inference of phylogeny and its impact on evol- taxonomic status of Gonocercinae Skrjabin et utionary biology. Science. 294:2310–2314. doi:10.1126/ Guschanskaja, 1955. Parasitology International. 67:74–78. science.1065889. doi:10.1016/j.parint.2017.03.007. Korotaeva VD. 1976. Trematodes from the family Sokolov SG, Atopkin DM, Urabe M, Gordeev II. 2019. Accacoeliidae in Pacific Ocean fish. Biologiya Morya. Phylogenetic analysis of the superfamily Hemiuroidea 4:60–61 (In Russian). (Platyhelminthes, Neodermata: Trematoda) based on Kumar S, Stecher G, Tamura K. 2016. MEGA7: molecular evol- partial 28S rDNA sequences. Parasitology. 146:596–603. utionary genetics analysis version 7.0 for bigger datasets. doi:10.1017/S0031182018001841. Molecular Biology and Evolution. 33:1870–1874. doi:10. Tkach VV, Littlewood DTJ, Olson PD, Kinsella JM, Swiderski Z. 1093/molbev/msw054. 2003. Molecular phylogenetic analysis of the 10 S. G. SOKOLOV ET AL.

Microphalloidea Ward, 1901 (Trematoda: Digenea). Vidal-Martínez VM, Velázquez-Abunader I, Centeno-Chalé Systematic Parasitology. 56:1–15. doi:10.1023/ OA, May-Tec AL, Soler-Jimenez LC, Pech D, Marino-Tapia A:1025546001611. I, Enriquez C, Zapata-Perez O, Herrera-Silveira J, et al. Truett GE. 2006. Preparation of genomic DNA from 2019. Metazoan parasite infracommunities of the dusky tissues. In: Kieleczawa J, editor. The DNA flounder (Syacium papillosum) as bioindicators of environ- book: protocols and procedures for the modern mol- mental conditions in the continental shelf of the Yucatan ecular biology. Sudbury: Jones & Bartlett Publisher; Peninsula, Mexico. Parasites & Vectors. 12:277. doi:10. p. 33–46. 1186/s13071-019-3524-6.