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Zoological Journal of the Linnean Society, 2020, XX, 1–19. With 5 figures. Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlaa093/5902681 by Federal Scientific Center of the East Asia user on 14 September 2020

An opisthorchiid concept of the Liliatrema (: : Opisthorchioidea): an unexpected systematic position

SERGEY SOKOLOV1, EVGENIY FROLOV2, SEMEN NOVOKRESHCHENNYKH2 and DMITRY ATOPKIN3*

1A. N. Severtsov Institute of Ecology and Evolution, Moscow, Russia 2Institute of Fisheries and Oceanography, Sakhalin branch (SakhNIRO), Yuzhno-Sakhalinsk, Russia 3Federal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch of the RAS, Vladivostok, Russia

Received 3 May 2020; revised 19 June 2020; accepted for publication 15 July 2020

Liliatrema is a small genus of trematodes consisting of two . Its systematic position has long been debated, partly because of the confusing reports about the structure of male terminal genitalia. Here we test the phylogenetic position of the genus Liliatrema using data on complete 18S rRNA and partial 28S rRNA gene sequences obtained for Liliatrema skrjabini. We also provide a detailed description of terminal genitalia in adult specimens of L. sobolevi and metacercariae of both Liliatrema species. The results of the 28S rDNA-based phylogenetic analysis indicate that Liliatrema falls within a well-supported clade, which also includes Apophallus and traditional opisthorchiids. This clade, in turn, is nested within a well-supported clade, containing Euryhelmis, and Scaphanocephalus. In the 18S+8S rDNA analysis, Liliatrema appears as a sister-taxon to the Cryptocotyle + Euryhelmis group. The Liliatrema + (Cryptocotyle + Euryhelmis) clade is a well-supported sister-group to the traditional opisthorchiids. The morphology of the terminal genitalia of the liliatrematids also corresponds to that of the opisthorchioids. Thus, the results of our morphological and phylogenetic analyses favour an unexpected conclusion that the genus Liliatrema belongs to the Opisthorchioidea. We propose that the genera Liliatrema, Apophallus, Euryhelmis, Cryptocotyle and Scaphanocephalus belong, respectively, within the subfamilies Liliatrematinae, Apophallinae, Euryhelminthinae and Cryptocotylinae of the family .

ADDITIONAL KEYWORDS: 18S – 28S – Apophallinae – Cryptocotylinae – Euryhelminthinae – Lepocreadioidea – Liliatrematidae – Opisthorchiidae.

INTRODUCTION 1953; Zhukov, 1960; Leonov et al., 1965; Machida, 1966; Belogurov et al., 1968; Ohbayshi & Araki, 1974; Liliatrema Gubanov, 1953 is a small genus of Wakabayashi, 1997; Frolov, 2010). Yamaguti (1958) has trematodes, comprising two described species: suggested that Liliatrema could be subdivided into two Liliatrema skrjabini Gubanov, 1953 (type species) and subgenera, Liliatrema Gubanov, 1953 (L. skrjabini) Liliatrema sobolevi Gubanov, 1953. The species of this аnd Liliatrematoides Yamaguti, 1958 (L. sobolevi), but genus implement their life cycle using marine other authors disagreed with this point of view (see: as second intermediate hosts and piscivorous Mehra, 1963; Skrjabin & Koval, 1966; Kostadinova as final hosts (Gubanov, 1953; Ohbayashi & Konno, & Gibson, 2005). The most striking morphological 1966; Belogurov et al., 1968; Ohbayshi & Araki, 1974; feature of this genus is a funnel-shaped oral Skorobrekhova, 2009). Their first intermediate hosts with a penta- or heptagonal distal (outer) end and a are unknown. These parasites are distributed in coastal dorsal edge that is covered with a hood-like tegumental water areas of the north-western Pacific (Gubanov, fold (Gubanov, 1953; Araki & Machida, 1990). The data on the structure of terminal genitalia in *Corresponding author. E-mail: [email protected] Liliatrema spp. were provided in five publications

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(Gubanov, 1953; Machida, 1966; Ohbayashi & Konno, extracted from the bodies using needles and a thick 1966; Araki & Machida, 1990; Kostadinova & Gibson, sagittal section of the body made with a sharp scalpel. 2005). In four of them, the presence of a well-developed All measurements were made in micrometres. cirrus-sac and an external seminal vesicle was reported Two specimens of each species were prepared for (Gubanov, 1953; Machida, 1966; Ohbayashi & Konno, SEM study by dehydration through a graded ethanol 1966; Kostadinova & Gibson, 2005). An accurate series and acetone followed by critical point drying. description of the proximal part of male terminal After coating with gold, they were examined with a genitalia in metacercaria of L. sobolevi was provided Tescan Vega TS5130MM microscope (s.r.o. TESCAN, by Araki & Machida (1990). These authors did not Czech Republic). Metacercariae of L. skrjabini for the find a cirrus-sac but found a bipartite seminal vesicle, molecular analysis were fixed in 96% ethanol and which lay free in the parenchyma. Unfortunately, the stored at –18 °C. morphological data of Araki & Machida (1990) were In addition, we studied an adult voucher specimen of not taken into account by the other authors. L. sobolevi (# 1274) ex Phalacrocorax pelagicus Pallas, The systematic position of the Liliatrema has been 1811, from the Penzhinsky District (Kamchatka Krai, repeatedly discussed, ecological and/or morphological Russia) deposited in the Museum of Helminthological evidence being used for taxonomical conclusions. Collections, A. N. Severtsov Institute of Ecology and This genus was originally placed in the subfamily Evolution, Russian Academy of Sciences, Moscow, Liliatrematinae Gubanov, 1953 of the family Russia (IPEE RAS). (see: Gubanov, 1953). Yamaguti (1958) moved Liliatrematinae to the family Cathaemasiidae. Mehra (1963), elevated the Liliatrematinae to DNA extraction, amplification and sequencing family status and included Liliatrematidae in the Total DNA was extracted from two metacercariae of Echinostomoidea. Skrjabin & Koval (1966), who were L. skrjabini using a ‘hot shot’ technique (Truett, 2006). unaware of Mehra’s work, also elevated Liliatrematinae Nuclear 18S rDNA, ITS1-5.8S-ITS2 rDNA and 28S rDNA to family status but placed it in Allocreadioidea. fragments were successfully amplified using polymerase Kostadinova & Gibson (2005) disagreed with all the chain reaction. Then 18S rDNA was amplified with the previous opinions on the systematic position of the following primers: Worm-A (5’ GCG AAT GGC TCA TTA liliatrematids and included these trematodes in the AAT CAG 3’) and 18S-F (5’ CCA GCT TGA TCC TTC TGC superfamily Lepocreadioidea. Bray et al. (2009) and AGG TTC ACC TAC 3’), described earlier (Littlewood Bray & Cribb (2012), revising the Lepocreadioidea, & Olson, 2001). Initial PCR reaction was performed in maintained liliatrematids in this superfamily. a total volume of 20 µL containing 0.25 mmol/L of each In this paper, we analyse the complete 18S rRNA and primer pair, 25 ng of total DNA in water, 5× Taq buffer, partial 28S rRNA gene sequences of metacercariae of 1.25 mmol/L dNTPs, 1.5 mmol/L magnesium and 1 unit L. skrjabini from coastal fish caught off Sakhalin Island. of Taq polymerase. Amplification of a 2000-bp fragment In addition, we provide new data on the morphology of of 18S rRNA gene was performed in a GeneAmp 9700, male terminal genitalia in both species of Liliatrema. Applied Biosystems, with a 5-min denaturation at 96 °C, Our data provide an unexpected solution to the problem 35 cycles of 1 min at 96 °C, 20 s at 58 °C and 5 min at of the familial and superfamilial affiliation of this genus. 72 °C and a 10-min extension at 72 °C. Negative and positive controls were made with the use of both primers. The ribosomal ITS1-5.8S-ITS2 fragment was MATERIAL AND METHODS amplified with the primers ITSF (5’-CGC CCG TCG CTA CTA CCG ATT G-3′) (Andres et al., 2014) Sample collection and S4R (5’-TAT GCT TAA ATT CAG CGG GT-3′) Encysted metacercariae of L. skrjabini and L. sobolevi (Besprozvannykh et al., 2019), with an annealing were collected from the musculature of Pholidapus temperature of 54 °C. dybowskii (Steindachner, 1880) (Actinopterygii: 28S ribosomal DNA (rDNA) was amplified with the Stichaeidae) and Hexagrammos octogrammus (Pallas, primers DIG12 (5’-AAG CAT ATC ACT AAG CGG-3′) 1814) (Actinopterygii: Hexagrammidae), respectively. and 1500R (5’-GCT ATC CTG AGG GAA ACT TCG-3′) The fish were caught at a depth of 3–5 m in the (Tkach et al., 2003), with an annealing temperature of coastal area of the southern part of Sakhalin Island 55 °C. PCR products were directly sequenced using an (46°58’35”N; 143°04’52”E). ABI Big Dye Terminator v.3.1 Cycle Sequencing Kit Metacercariae collected for morphological study were (Applied Biosystems, USA), as recommended by the taken from the cysts and fixed in hot 70% ethanol, manufacturer, with the internal sequencing primers stained with acetocarmine and mounted in Canadian described by Tkach et al. (2003) for 28S rDNA. PCR balsam. For a detailed morphological study of L. sobolevi, product sequences were analysed using an ABI 3130 we prepared two slides of isolated reproductive organs genetic analyser at the Federal Scientific Center of the

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East Asia Terrestrial Biodiversity FEB RAS. Sequences Specimens deposited: Three voucher specimens, IPEE were submitted to the GenBank database (NCBI). RAS 14286.

Representative DNA sequences: Two complete 18S Alignments and phylogenetic analysis rRNA gene sequences (MT303881 and MT303882), two Ribosomal DNA sequences were assembled with ribosomal ITS1 fragments (MT624319 and MT624320) SeqScape v.2.6 software, provided by Applied Biosystems. and two partial 28S rRNA gene sequences (MT303944 Alignments and estimations of the number of variable and MT303945), 1981 bp and 2641 bp (1138 bp used sites and sequence differences were performed using the for phylogenetic analysis) in length, respectively. MEGA 7.0 software (Kumar et al., 2016). Phylogenetic There were no variable sites within either 18S rDNA, relationships were obtained using a concatenated dataset 28S rDNA or ITS1 rDNA. of complete 18S rRNA gene and partial sequences of 28S rRNA gene, and using partial sequences of 28S rRNA Description (based on three whole-mounted excysted gene only. Phylogenetic analysis was performed using specimens and two SEM-studied specimens): Body the maximum likelihood and Bayesian algorithms elongate; length 2475–2941, maximum width 636–665 with the PhyML v.3.1 (Guindon & Gascuel, 2003) and in anterior half of body. spinose; spines MrBayes v.3.1.2 (Huelsenbeck et al., 2001) software, multipointed, becoming sparser toward posterior end respectively. The best nucleotide substitution models, the of body. Eye-spot remnants at level of pharynx. Oral TVM+I+G and GTR+I+G (Posada, 2003) were estimated sucker terminal, funnel-shaped, 539–630 × 483–553, with jModelTest v.2.1.5 software (Darriba et al., 2012) with pentagonal distal (outer) end. Dorsal edge of for 28S rDNA and concatenated 18S+28S rDNA distal end of oral sucker covered with hood-like fold datasets, respectively. Bayesian analysis was performed of tegument. Ventral sucker well developed, rounded, using 10 000 000 generations with two independent 203–214 × 207–210, recessed within tegumental crypt. runs. Summary parameters and the phylogenetic tree Forebody 30.3–33.9% of body length. Prepharynx were calculated with a burn-in of 25% of generations. 119–154. Pharynx oval 161 × 105–112. Oesophagus The significance of the phylogenetic relationships was 84–140. Intestine bifurcating distinctly anteriorly to estimated using posterior probabilities (Huelsenbeck ventral sucker. Caeca reaching posterior end of body, et al., 2001). GenBank sequence data for representatives connecting to excretory vesicle, forming uroproct. of Apocreadioidea, Brachycladioidea, Gorgoderoidea, Testes two, rounded to suboval, entire or slightly Haploporoidea, Lepocreadioidea, Monorchioidea, indented, tandem, in posterior half of body; anterior Opecoeloidea, Opisthorchioidea, and Echinostomatoidea testis 259–301 × 329–371, posterior testis 364 × 280– (as outgroup taxa) used in molecular analysis, including 315. Post-testicular region 18.8–21.7% of body length. references and accession numbers, are given in Table 1. Cirrus-sac absent. Seminal vesicle, pars prostatica Though the genus Pachytrema Looss, 1907 is included with field of prostatic cells, and ejaculatory duct in the system of the Opisthorchiidae by Scholz (2008) lying free in parenchyma. Seminal vesicle bipartite, within the subfamily Pachytrematinae Railliet, 1919, with relatively short proximal portion and long Pérez-Ponce de León & Hernández-Mena (2019) have distal portion, running dextral to ventral sucker. shown that Pachytrema probably does not belong to Pars prostatica vesicular, surrounded by field of the Opisthorchioidea. Therefore, we do not consider this prostatic cells. Ejaculatory duct short, connecting genus in our study. with genital atrium. Genital atrium short, opening dextro-submedially at base of genital sac. Genital sac with lip-like posterior wall overlapping anterior part of recessed ventral sucker; median. RESULTS Ovary trilobed, pretesticular and dextrosubmedial, Morphology 140–154 × 147–175. Seminal receptacle canalicular, Metacercaria of Liliatrema skrjabini saccular, mainly posterior to ovary. Laurer’s canal Gubanov, 1953 opening dorsomedially just posterior to ovary level. Oötype not observed. Mehlis’ gland extensive. Uterus (Figs 1A, 2A, B) pretesticular, running sinistrally to a ventral sucker, curving anteriorly to the sucker and opening in the Host: Pholidapus dybowskii. genital atrium. Vitellarium follicular; anlage of follicles in two longitudinal lateral fields extending from level Locality: The north-west Pacific off the southern part of pharynx to posterior extremity of body, overlapping of Sakhalin Island. caeca ventrally and dorsally, and confluent in the post- testicular region. Excretory vesicle Y- to T-shaped, Site of infection: Musculature. extending to seminal receptacle.

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Table 1. List of previously published sequences used in the phylogenetic analysis

Species 18S rRNA gene 28S rRNA gene Reference

Apocreadioidea Homalometron armatum (MacCallum, 1895) AY222130 AY222241 Olson et al. (2003) Homalometron synagris (Yamaguti, 1953) AJ287523 – Cribb et al. (2001) – AY222243 Olson et al. (2003) Neoapocreadium splendens Cribb & Bray, 1999 AJ287543 – Cribb et al. (2001) – AY222242 Olson et al. (2003) Schistorchis zancli Hanson, 1953 AY222129 AY222240 Olson et al. (2003) Brachycladioidea Pleorchis polyorchis (Stossich, 1889) DQ248202 DQ248215 Bray et al. (2005) Pleorchis uku Yamaguti, 1970 DQ248203 DQ248216 Bray et al. (2005) (Nicoll, 1907) DQ248205 DQ248218 Bray et al. (2005) Stephanostomum bicoronatum (Stossich, 1883) DQ248212 DQ248225 Bray et al. (2005) Stephanostomum cesticillus (Molin, 1858) DQ248213 DQ248226 Bray et al. (2005) Stephanostomum interruptum Sparks & Thatcher, 1958 DQ248210 DQ248223 Bray et al. (2005) Stephanostomum gaidropsari Bartoli & Bray, 2001 DQ248208 DQ248221 Bray et al. (2005) Stephanostomum minutum (Looss, 1901) DQ248211 DQ248224 Bray et al. (2005) Stephanostomum pristis (Deslongchamps, 1824) DQ248209 DQ248222 Bray et al. (2005) Stephanostomum tantabiddii Bray & Cribb, 2004 DQ248207 DQ248220 Bray et al. (2005) Tormopsolus orientalis Yamaguti, 1934 DQ248204 DQ248217 Bray et al. (2005) Brachycladium goliath (Van Beneden, 1858) KR703279 KR703279 Briscoe et al. (2016) Zalophotrema hepaticum Stunkard & Alvey, 1929 AJ224884 – Cribb et al. (2001) – AY222255 Olson et al. (2003) Gorgoderoidea Allocreadiidae neotenicum Peters, 1957 JX983204 JX977132 Bray et al. (2012) Brachylecithum lobatum (Railliet, 1900) AY222144 AY222260 Olson et al. (2003) (Rudolphi 1819) Y11236 – Sandoval (unpublished) – AF151939 Tkach et al. (2000) Lyperosomum collurionis (Skrjabin & Isaichikov, 1927) AY222143 AY222259 Olson et al. (2003) Degeneria halosauri (Bell, 1887) AJ287497 – Cribb et al. (2001) – AY222257 Olson et al. (2003) Gorgodera sp. AJ287518 – Cribb et al. (2001) – AY222264 Olson et al. (2003) Nagmia floridensis Markell, 1953 AY222145 AY222262 Olson et al. (2003) Haploporoidea Atractotrematidae Atractotrema sigani Durio & Manter, 1969 AJ287479 – Cribb et al. (2001) – AY222267 Olson et al. (2003) Pseudomegasolena ishigakiense Machida & Kamiya, AJ287569 – Cribb et al. (2001) 1976 – AY222266 Olson et al. (2003) Elonginurus mugilus Lü, 1995 – MH763761 Atopkin et al. (2019) MN718679 – Sokolov et al. (2020) Hapladena nasonis Yamaguti, 1970 AY222146 AY222265 Olson et al. (2003) Haploporus benedeni (Stossich, 1887) FJ211228 FJ211237 Blasco-Costa et al. (2009)

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Table 1. Continued

Species 18S rRNA gene 28S rRNA gene Reference

Lepocreadioidea Aephnidiogenidae Tetracerasta blepta Watson, 1984 L06670 – Blair & Baker (1993) – FJ788494 Bray et al. (2009) Gorgocephalidae Gorgocephalus kyphosi Manter, 1966 AY222126 AY222234 Olson et al. (2003) Paragyliauchen arusettae Machida, 1984 AY222127 – Olson et al. (2003) – FJ788503 Bray et al. (2009) Enenterum aureum Linton, 1910 AY222124 AY222232 Olson et al. (2003) Koseiria xishaensis Gu & Shen, 1983 AY222125 AY222233 Olson et al. (2003) Preptetos caballeroi Pritchard, 1960 AJ287563 – Cribb et al. (2001) – AY222236 Olson et al. (2003) Preptetos trulla (Linton, 1907) AY222128 AY222237 Olson et al. (2003) Opecoeloidea Halosaurotrema halosauropsi (Bray & Campbell, AJ287514 – Cribb et al. (2001) 1996) [access as Gaevskajatrema halosauropsi] – AY222207 Olson et al. (2003) Macvicaria macassarensis (Yamaguti, 1952) AJ287533 – Cribb et al. (2001) – AY222208 Olson et al. (2003) idoneum (Nicoll, 1909) AJ287558 – Cribb et al. (2001) – AY222209 Olson et al. (2003) Monorchioidea Lissorchis kritskyi Barnhart & Powell, 1979 AY222136 AY222250 Olson et al. (2003) Cableia pudica Bray, Cribb & Barker, 1996 AJ287486 – Cribb et al. (2001) – AY222251 Olson et al. (2003) Diplomonorchis leiostomi Hopkins, 1941 AY222137 AY222252 Olson et al. (2003) Lasiotocus typicum (Nicoll, 1912) [access as AJ287474 – Cribb et al. (2001) Ancylocoelium typicum] – AY222254 Olson et al. (2003) Provitellus turrum Dove & Cribb, 1998 AJ287566 – Cribb et al. (2001) – AY222253 Olson et al. (2003) Opisthorchioidea Acanthostomum burminis (Bhalerao, 1926) – KC489791 Jayawardena et al. (2013) Acanthostomum minimum Stunkard, 1938 – MK648271 Pérez-Ponce de Leon & Hernández-Mena (2019) Adlardia novaecaledoniae Miller et al., 2009 – FJ788496 Bray et al. (2009) Caecincola parvulus Marshal & Gilbert, 1905 AY222123 AY222231 Olson et al. (2003) Mitotrema anthostomatum Manter, 1963 AJ287542 – Cribb et al. (2001) – AY222229 Olson et al. (2003) Neocladocystis bemba Georgieva, Kmentová & Bray, – MN705808 Kmentová et al. (2020) 2020 Neocladocystis biliaris Georgieva, Kmentová & Bray, – MN705809 Kmentová et al. (2020) 2020 Siphodera vinaledwardsii (Linton, 1901) AY222122 AY222230 Olson et al. (2003)

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Table 1. Continued

Species 18S rRNA gene 28S rRNA gene Reference

Tabascotrema verai Lamothe-Argumedo & Pineda- – MK648275 Pérez-Ponce de Leon López, 1990 & Hernández-Mena (2019) Tanganyikatrema fusiforma Kmentová et al., 2020 – MN705811 Kmentová et al. (2020) Timoniella imbutiforme (Molin, 1859) – MF491865 Kvach et al. (2018) Ascocotyle cameliae Hernández-Orts et al., 2019 – MK359080 Hernández-Orts et al. (2019) Ascocotyle longa Ransom, 1920 MF980222 MF980611 Santos & Borges (unpub- lished) Ascocotyle patagoniensis Hernández-Orts et al., 2012 – MK359082 Hernández-Orts et al. (2019) (Nishigori, 1924) HQ874608 HQ874609 Thaenkham et al. (un- published) bearupi Pearson, 1973 – MH257773 Huston et al. (2018) Galactosomum lacteum (Jägerskiöld, 1896) AY222120 AY222227 Olson et al. (2003) Galactosomum ubelakeri (Dailey, 1969) – MG806920 Kuzmina et al. (2018) pumilio (Looss, 1896) KX815125 KX815125 Le et al. (2017) (Nishigori, 1924) KX815126 KX815126 Le et al. (2017) Haplorchis yokogawai (Katsuta, 1932) HM004207 HM004178 Thaenkham et al. (2010) Haplorchoides daguilarensis Hostettler et al., 2018 – MG747499 Hostettler et al. (2018) Haplorchoides maiwariensis Hostettler et al., 2018 – MG747501 Hostettler et al. (2018) Metagonimoides oregonensis Price, 1931 – JQ995473 Belden et al. (2012) miyatai Saito et al., 1997 HQ832626 HQ832635 Pornruseetairatn et al. (2016) Metagonimus pusillus Tatonova et al., 2018 – MF407173 Tatonova et al. (2018) Metagonimus suifunensis Shumenko et al., 2017 – KX387460 Shumenko et al. (2017) Suzuki, 1930 HQ832629 HQ832638 Pornruseetairatn et al. (2016) (Katsurada, 1912) HQ832632 HQ832641 Pornruseetairatn et al. (2016) Phocitrema fusiforme Goto & Ozaki, 1930 – MG806921 Kuzmina et al. (2018) Procerovum cheni Hsü, 1950 HM004212 HM004193 Thaenkham et al. (2010) Procerovum varium Onji & Nishio, 1916 MF077365 Lee et al. (unpublished) – KY369161 Lee et al. (2017) Pygidiopsis macrostomum Travassos, 1928 MF972492 MF972531 Santos & Borges (unpub- lished) falcatus Onji & Nishio, 1916 HM004202 HM004174 Thaenkham et al. (2010) Opisthorchiidae Apophallinae Apophallus zalophi Price, 1932 – MG806918 Kuzmina et al. (2018) Cryptocotylinae Cryptocotyle lata Tatonova & Besprozvannykh, 2019 – MH025623 Tatonova & Besprozvannykh (2019) Cryptocotyle lingua (Creplin, 1825) AJ287492 – Cribb et al. (2001) – AY222228 Olson et al. (2003) Scaphanocephalus expansus (Creplin, 1842) – MK680936 Kohl et al. (2019) Euryhelminthinae Euryhelmis costaricensis Brenes-Madrigal et al., 1960 AB521799 AB521799 Sato et al. (2010) Metorchiinae Metorchis orientalis Tanabe, 1921 MK482055 MK482055 Qiu et al. (2020)

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Table 1. Continued

Species 18S rRNA gene 28S rRNA gene Reference

Metorchis ussuriensis Besprozvannykh et al., 2019 – KY075772 Besprozvannykh et al. (2019) Opisthorchiinae Amphimerus ovalis Barker, 1911 AY222121 AY116876 Olson et al. (2003) (Cobbold, 1875) MK450525 MK450525 Qiu et al. (2020) Erschoviorchis anuiensis Tatonova et al., 2020 – MK877245 Tatonova et al. (2020) felineus (Rivolta, 1884) MF077357 MF099790 Dao et al. (2017) (Poirier, 1886) JF823987 JF823990 Thaenkham et al. (2011) Outgroup Isthmiophora hortensis (Asada, 1926) AB189982 AB189982 Sato & Suzuki (2006) gralli (Mathis & Leger, 1910) JQ627832 JQ627832 Literák et al. (2013)

Metacercaria of Liliatrema sobolevi short proximal portion and long distal portion, running Gubanov, 1953 dextrally to ventral sucker. Pars prostatica vesicular, (Figs 1B, 2С–E, 3A) surrounded by field of prostatic cells. Ejaculatory duct short, connecting with genital atrium. Genital Host: Hexagrammos octogrammus. atrium short, opening dextro-submedially at base of genital sac. Genital sac with lip-like posterior wall Locality: The north-west Pacific off the southern part overlapping anterior part of recessed ventral sucker; of Sakhalin Island. median, surrounded by smaller gland cells. Ovary tetralobed, pretesticular and dextrosubmedial, 105–126 × 189–196. Seminal receptacle canalicular, Site of infection: Musculature. saccular, mainly posterior to ovary. Laurer’s canal opening dorsomedially just posterior to ovary level. Oötype not Specimens deposited: Three voucher specimens, IPEE observed. Mehlis’ gland extensive. Uterus pretesticular, RAS 14287, 14288. running sinistrally to ventral sucker, curved anteriorly to sucker and opening in genital atrium. Vitellarium Description (based on three whole-mounted excysted follicular; anlage of follicles in two longitudinal lateral specimens, two SEM-studied specimens, two slides with fields extending from level of ovary to posterior extremity the isolated terminal genitalia and one slide with the of body, overlapping caeca ventrally and dorsally, and sagittal section of the body): Body elongate; length 4497– almost confluent in post-testicular region. Excretory 5013, maximum width 976–1061 in posterior half of vesicle Y-shaped, extending to ovary level. body. Tegument spinose; spines multipointed, becoming sparser toward posterior end of body. Eye-spots at the level of the prepharynx. Oral sucker terminal, funnel- Male terminal genitalia of adult of Liliatrema shaped, 650–707 × 763–820, with heptagonal distal sobolevi (outer) end. Dorsal edge of distal end of oral sucker (Fig. 3B) covered with hood-like fold of tegument. Ventral sucker well developed, rounded, 329–357 × 294–315, recessed within tegumental crypt. Forebody 45.6–50.9% of Host: Phalacrocorax pelagicus. body length. Prepharynx 196–350. Pharynx elongate- oval 378–385 × 49–112. Oesophagus 280. Intestine Locality: Penzhinsky District, Kamchatka Krai, bifurcating distinctly anteriorly to ventral sucker. Russia. Caeca reaching to posterior end of body, connecting to excretory vesicle, forming uroproct. Site of infection: Intestines. Testes two, transversely elongated, indented, nearly tandem, in posterior half of body; anterior testis 140– Specimens deposited: Voucher specimen, IPEE RAS 224 × 455–462, posterior testis 182–245 × 385–434. 1274. Post-testicular region 19.5–20.8% of body length. Cirrus-sac absent. Seminal vesicle, pars prostatica with Description (based on one specimen): Cirrus-sac field of prostatic cells, and ejaculatory duct lying free in absent. Seminal vesicle, pars prostatica with field parenchyma. Seminal vesicle bipartite, with relatively of prostatic cells, and ejaculatory duct lying free in

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Figure 1. Metacercariae of Liliatrema spp., whole view. A, L. skrjabini. B, L. sobolevi. Scale bars: 1 mm. parenchyma. Seminal vesicle large, 298 in length, Ejaculatory duct short, muscular, connecting with bipartite, with relatively short proximal portion and genital atrium. Genital atrium short, opening dextro- long distal portion, terminating in small sphincter, submedially at base of genital sac. Genital sac with running dextrally to ventral sucker. Pars prostatica lip-like posterior wall overlapping anterior part of vesicular, surrounded by field of prostatic cells. recessed ventral sucker; median.

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Figure 2. SEM microphotographs of Liliatrema spp. A, oral sucker of L. skrjabini. B, ventral sucker and genital sac of L. skrjabini. C, oral sucker of L. sobolevi. D, multipointed tegumental spines of L. sobolevi. E, ventral sucker and genital sac of L. sobolevi. Scale bars: A, C, 0.2 mm; B, 0.05 mm; D, 0.01 mm; E 0.1 mm.

Phylogenetic data opisthorchiid genera (Fig. 4А). The Opisthorchiinae The 28S-based maximum likelihood (ML) and + Metorchiinae clade shows a distinct monophyly. Bayesian (BI) phylogenetic trees (Fig. 4A, B) However, the subfamily Opisthorchiinae, which is demonstrate that Liliatrema falls within a large well- represented in our study by the genera Amphimerus, supported clade, which also includes opisthorchiine Clonorchis, Erschoviorchis and Opisthorchis, appears genera Amphimerus Barker, 1911, Clonorchis Loos, to be paraphyletic (Fig. 4А, B). Another opisthorchiid 1907, Erschoviorchis Skrjabin, 1945 and Opisthorchis (sensu Tatonova & Besprozvannykh, 2019) genus, Blanchard, 1895, a metorchiine genus Metorchis Cryptocotyle Lühe, 1899, reliably clustered with the Looss, 1899 and the opisthorchiid (sensu Tatonova heterophyid genus Scaphanocephalus Jägerskiöld, & Besprozvannykh, 2019) genus Apophallus Lühe, 1903. The phylogenetic position of the Cryptocotyle + 1909. Within this clade, Liliatrema appears as a Scaphanocephalus group is labile, depending on the sister-taxon to the poorly supported Apophallus + phylogenetic algorithm used. This group appears as (Opisthorchiinae + Metorchiinae) group on the BI tree a sister-group to the opisthorchiid (sensu Tatonova & (Fig. 4B), but forms a poorly supported group with Besprozvannykh, 2019) genus Euryhelmis Poche, 1926, Apophallus in the ML tree. The latter group occupies with poor support on the BI tree (Fig. 4B). In turn, a sister-position in relation to the clade of traditional the Euryhelmis + (Cryptocotyle + Scaphanocephalus)

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Figure 3. Liliatrema sobolevi. A, sagittal section of the body of metacercaria in ventral sucker region; B, terminal genitalia, ventral sucker and genital sac in adult specimen. Abbreviations: ej, ejaculatory duct; ga, genital atrium; gc, gland cells surrounding genital sac; gp, genital pore; gs, genital sac; mt, metraterm; mtu, distal part of female duct (uterine loops and metraterm); pl, lip-like posterior wall of genital sac; pp, pars prostatica, sv, bipartite vesicle; vs, ventral sucker. Scale bars: A, 0.5 mm; B, 0.15 mm. and the Liliatrema + [Apophallus + (Opisthorchiinae also contains monophyletic group of the traditional + Metorchiinae)] clades are sister-groups. On the opisthorchiid genera Clonorchis, Opisthorchis, ML tree, the Cryptocotyle + Scaphanocephalus Metorchis and Amphimerus (Fig. 5A, B). The group appears as a sister-taxon to the poorly Cryptocotyle + Euryhelmis group has high support supported Euryhelmis + [(Liliatrema + Apophallus) + on BI phylogenetic tree and moderate support on ML (Opisthorchiinae + Metorchiinae)] clade (Fig. 4A). The tree. The Opisthorchiinae appears as a paraphyletic heterophyid genus Metagonimoides Price, 1931 has a group. Heterophyid species form two early-branching basal position to all the trematodes mentioned above, groups, which are united into a weakly supported but it is supported only in the ML tree (Fig. 4A, B). clade. However, only one of these groups, the one All the other heterophyids are distributed across three that contains the genera Galactosomum, Haplorchis, early-branching groups, but only one of these groups, Metagonimus, Procerovum and Stellantchasmus, is the one containing the genera Galactosomum Looss, highly supported. The phylogenetic position of the 1899, Haplorchis Looss, 1899, Haplorchoides Chen, Cryptogonimidae corresponds to that on the 28S-based 1949, Metagonimus Katsurada, 1912, Procerovum trees (Fig. 4A). Onji & Nishio, 1916 and Stellantchasmus Onji & Two variants of an opisthorchiid-specific 30-bp Nishio, has statistically significant supports on both repeat within 361-bp ribosomal ITS1 were detected ML and BI trees. Species of the Cryptogonimidae for L. skrjabini in positions 67–96 bp and 154–183 bp, form a distinct clade, which is basal to the group respectively. The first repeat sequence variant is including traditional opisthorchiids and the genera characterized by a unique nucleotide composition, Apophallus, Cryptocotyle, Euryhelmis, Liliatrema and containing a CCC triplet at positions 8–10. Scaphanocephalus (Fig. 4A). Phylogenetic ML and BI analyses, based on concatenated complete 18S and partial 28S rRNA gene sequences, showed that Liliatrema is closely DISCUSSION related to the Cryptocotyle + Euryhelmis group We provide a detailed description of terminal genitalia within a common highly supported clade, which in adult specimens of L. sobolevi and metacercariae of

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Figure 4. Phylogenetic position of Liliatrema based on dataset of partial 28S rRNA gene sequences alignment. A, reconstruction with maximum likelihood algorithm using Bayesian nodal support; B, alternative topology of the Opisthorchiidae in Bayesian algorithm. The posterior probabilities less than 0.9 are not indicated. References for data retrieved from GenBank are listed in Table 1.

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Figure 5. Phylogenetic position of Liliatrema based on dataset of 18S+28S rRNA genes sequences alignment. A, reconstruction with maximum likelihood algorithm using Bayesian nodal support; B, fragment of tree with topology of the Opisthorchiidae in Bayesian algorithm. The posterior probabilities less than 0.9 are not indicated. References for data retrieved from GenBank are listed in Table 1.

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Based on our results, we conclude data, the presence of long repeats within their ITS1 that the structures in Liliatrema spp. referred to in the rDNA and the fact that all these trematodes use snails literature as pairs of apron-like projections (Machida, of the superfamily Truncatelloidea as first intermediate 1966; Ohbayshi & Araki, 1974; Araki & Machida, hosts. Unfortunately, these authors did not provide 1990) are a lateral thickening of the lip-like posterior an amended diagnosis of the Opisthorchiidae. We walls of the genital sac (Fig. 2B, E). We confirm the accept the taxonomic hypothesis of Tatonova & presence of canalicular seminal receptacles in the Besprozvannykh (2019) and expand the generic metacercariae of L. skrjabini and L. sobolevi. This composition of the Opisthorchiidae by the addition organ was described in adults of Liliatrema spp. (see: of Scaphanocephalus and Liliatrema on the basis of Gubanov, 1953; Machida, 1966), but not noted in its the current molecular phylogenetic data, the presence metacercariae (Ohbayashi & Konno, 1966; Ohbayshi & of opisthorchiid-specific 30-bp ITS1 rDNA repeats Araki, 1974; Araki & Machida, 1990). In other respects, in Liliatrema (its presence in Scaphanocephalus our original morphological data for the metacercariae is yet unproven) and a similar morphology of the of L. skrjabini and L. sobolevi agree with the literature ventrogenital complex in Scaphanocephalus and data (Ohbayashi & Konno, 1966; Ohbayshi & Araki, Cryptocotyle. The relative localization of the two repeats 1974; Araki & Machida, 1990; Frolov, 2010). in Liliatrema is similar to that described by Tatonova The results of the phylogenetic analysis disprove & Besprozvannykh (2019) for opisthorchiines and earlier hypotheses about the superfamilial affiliation metorchiines. However, we would like to emphasize of the liliatrematids (see: Mehra, 1963; Skrjabin that the first repeat in Liliatrema is characterized by a & Koval, 1966; Kostadinova & Gibson, 2005) unique nucleotide composition, which was not detected and indicate that they belong to the superfamily in any of the opisthorchiid species. The nucleotide Opisthorchioidea. The morphology of the terminal composition of the second repeat sequence variant genitalia of liliatrematids also corresponds to that of corresponds to that of Apophallus donicus (Skrjabin the opisthorchioids (e.g. Bray, 2008). & Lindtrop, 1919), Cryptocotyle lata Tatonova & The results of the phylogenetic analyses, based Besprozvannykh, 2019, Cryptocotyle lingua (Creplin, on the concatenated complete 18S and partial 28S 1825) and Euryhelmis costaricensis Brenes-Madrigal rRNA gene sequence data, show that traditional et al., 1960 (see Tatonova & Besprozvannykh, 2019). opisthorchiids and the genera Liliatrema, Cryptocotyle The phylogenetic tree topology for Apophallus, and Euryhelmis have a common nearest ancestor Cryptocotyle, Euryhelmis, Liliatrema, (Fig. 5A, B). An additional analysis based on 28S Scaphanocephalus and the traditional opisthorchiids rRNA gene sequences indicates that the clade, which presented in our study, in combination with the unites the parasites listed above, also contains the morphology of the oral sucker, the caeca and the genera Apophallus and Scaphanocephalus (Fig. 4A, ventrogenital complex (if any) in representatives of B). Significantly, the position of Liliatrema within these groups (see: Pearson, 2008; Scholz, 2008; present the clade under discussion is labile, depending on the data), can be interpreted within the framework molecular dataset (28S rDNA or 18S+28S rDNA). of the taxonomic concept of the four subfamilies: The genus Apophallus was integrated into one group Apophallinae Ciurea, 1924, Cryptocotylinae with the traditional opisthorchiids and Liliatrema. Lühe, 1909, Euryhelminthinae Morosov, 1952 and However, the sister-taxon for Apophallus cannot yet Liliatrematinae Gubanov, 1953. be identified unambiguously. The genera Cryptocotyle The Apophallinae originally included only the genera and Scaphanocephalus constitute a monophyletic Apophallus and Rossicotrema Skrjabin, 1919 (see: group of trematodes, morphologically supported by the Ciurea, 1924). Later this subfamily was complemented similarity of the ventrogenital complex (see: Pearson, with Pricetrema Ciurea, 1933 and Euryhelmis (see: 2008). Both BI and ML tree topologies, based on Ciurea, 1933) and Tauridiana Isaichikov, 1925 and complete 18S and partial 28S rRNA gene sequences, Ponticotrema Isaichikov, 1927 (see: Price, 1940). testify that this group (more precisely, its single Morozov (1952) moved Euryhelmis to Euryhelminae representative on the 18S+28S rDNA-based trees) (correctly Euryhelminthinae), moved Tauridiana is closely related to the genus Euryhelmis. However, and Ponticotrema to the Knipowitschetrematinae the poor support of these relationships on 28S rDNA- Morozov, 1950 of Galactosomatidae Morozov, 1950 and based trees indicates that this conclusion needs to be left Apophallus, Pricetrema and Rossicotrema within verified. Apophallinae. The assignment of Euryhelmis to a

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Pearson (2008) did new synonyms Liliatrematidae Gubanov, 1953 and not use subfamilies in his system of heterophyids at Heterophyidae Leiper, 1909 pro parte. all. To date, it seems reasonable to consider the names Rossicotrema, Pricetrema and Apophalloides as junior Diagnosis [modified from family diagnosis of Scholz synonyms of Apophallus (e.g. Pearson, 2008; Sándor (2008) and our data]: Body small- to medium- et al., 2017). Pearson (2008) excluded the genera sized, variable in shape, unarmed or spined. Oral Ponticotrema and Tauridiana from the Heterophyidae, sucker terminal or subterminal, unspecialized or leaving them as incertae sedis. The genus Euryhelmis, funnel-shaped, with penta- or heptagonal distal end, as we have shown above with the help of the sometimes reduced or rudimentary. Ventral sucker phylogenetic analysis, is considerably separated from usually pre-equatorial, well developed, exceptionally Apophallus (Fig. 4A, B). reduced or rudimentary. Pharynx present. Oesophagus Cryptocotylinae was created by Lühe (1909) for the short or long. Intestine bifurcating in forebody. Caeca genera Cryptocotyle and Scaphanocephalus. Ciurea long, reaching testicular level or posterior extremity, (1933) added the genera Tocotrema Looss, 1899 straight or sinuous, blind, exceptionally opening via and Ciureana Skrjabin, 1923 into this subfamily. two ani or forming uroproct. Testes two or one, variable However, most of the subsequent authors recognized in shape, tandem, oblique or opposite, in posterior half the generic names Tocotrema and Ciureana as of body. Cirrus-sac absent. Seminal vesicle usually junior synonyms of Cryptocotyle (e. g. Price, 1940; tubular, undivided or bipartite, in forebody, rarely Yamaguti, 1958; Pearson, 2008). Price (1940) included equatorial or in hindbody. Genital or ventrogenital three genera, Cryptocotyle, Scaphanocephalus and sac absent, occasionally present; if present, median: Taphrogonymus Cohn, 1904, into this subfamily. genital sac without gonotyl, and ventrogenital Morozov (1952) simplified the structure of the sac containing one or two unarmed preacetabular Cryptocotylinae to two genera, Cryptocotyle and gonotyls and well-developed ventral sucker or without Ciureana, and defended the validity of the latter. gonotyl but with rudimentary ventral sucker. Genital Without any apparent reason, this author (Morozov, pore median or slightly submedian, mostly to ventral 1952) did not consider the genera Scaphanocephalus sucker. Ovary entire or lobed, median or submedian, and Taphrogonymus in Heterophyidae. According mostly pretesticular. Seminal receptacle canalicular, to Yamaguti (1958), the Cryptocotylinae contained rarely uterine. Uterus pretesticular, rarely posterior only the genera Cryptocotyle and Metagonimoides. to testes. Vitellarium follicular, in two lateral fields. He moved the genus Taphrogonymus Cohn, 1904 to Excretory vesicle Y- or T-shaped. Adults in liver, the subfamily Pygidiopsinae Yamaguti, 1958, and biliary system, pancreas or intestine of terrestrial assigned Scaphanocephalus to a separate subfamily and aquatic vertebrates, cosmopolitan. Type genus Scaphanocephalinae Yamaguti, 1958. Later, Yamaguti Opisthorchis Blanchard, 1895. (1971) added the genus Massaliatrema Dollfus & Timon-David, 1960 to Cryptocotylinae. According to Pearson (2008), Massaliatrema and Metagonimoides Subfamily Apophallinae ciurea, 1924 are congeneric with Cryptocotyle and Metagonimus, respectively. However, concerning Metagonimoides, Diagnosis [modified from subfamily diagnosis of the opinions of Pearson and that of S. Yamaguti (see Yamaguti,(1971) and generic diagnosis of Pearson above) are not supported with modern phylogenetic (2008)]: Body pyriform to elliptical or more elongate, results (Fig. 4A, B). spined. Oral sucker unspecialized. Ventral sucker Accepting the above taxonomical concepts (Lühe, well developed, with axis inclined anteriorly. Pharynx 1909; Ciurea, 1924, 1933; Price, 1940; Morozov, 1952; present. Caeca terminating at or near posterior Yamaguti, 1958, 1971; Pearson, 2008) and the current extremity. Testes two or one, entire, tandem or phylogenetic data, we consider that Apophallinae and oblique. Cirrus-sac absent. Seminal vesicle elongate, Euryhelminthinae are monogeneric taxa and that S-shaped, undivided but sometimes constricted; pars the Cryptocotylinae contains the genera Cryptocotyle prostatica present. Ventrogenital sac permanent, and Scaphanocephalus. Here, we provide amended median. Gonotyls two, lateral and opposite, arising diagnoses of the Opisthorchiidae and its subfamilies dorsally close against ventral sucker, clavate in

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Diagnosis [modified from subfamily diagnosis of Yamaguti,(1971) and generic diagnosis of Pearson Family Liliatrematinae Gubanov, 1953 emend. (2008)]: Body ovoid to pentagonal or elongate with wing-like anterolateral expansions, spined. Oral Syn.: Liliatrematinae Gubanov, 1953. sucker unspecialized. Ventral sucker rudimentary, on large protractile bulge of posterior wall of Diagnosis [modified from the Liliatrematidae diagnosis ventrogenital sac. Pharynx present. Caeca terminating of Kostadinova & Gibson (2005) and our data]: Body at or near posterior extremity. Testes two, lobed or elongate-fusiform, spined. Oral sucker terminal, entire, opposite, oblique or tandem. Cirrus-sac absent. funnel-shaped, with penta- or heptagonal distal Seminal vesicle elongate, undivided or bipartite; pars end. Dorsal edge of distal end of oral sucker covered prostatica present. Ventrogenital sac permanent, with hood-like fold of tegument. Ventral sucker well median, with prominent transverse band of muscle developed, rounded, recessed within tegumental crypt. fibres just anterior to mouth of ventrogenital sac, Pharynx present. Caeca reaching to posterior end of with anterior pocket lacking continuous capsule and body, connecting to excretory vesicle, forming uroproct. outer limiting membrane, with strong transverse Testes two, entire or indented, tandem or almost so. band of muscle fibres forming hemisphincter ventral Cirrus-sac absent. Seminal vesicle elongate, bipartite; to entrance of pocket into ventrogenital sac proper. pars prostatica present. Genital sac with thin anterior Gonotyl absent. Common genital pore or separate and thick lip-like posterior walls, overlapping anterior male and female genital pores dorsal to bulge. part of recessed ventral sucker. Gonotyl absent. Ovary lobed or entire, median or submedian, pre- or Genital pore slightly dextro-submedian, anterior to intertesticular. Seminal receptacle canalicular. Uterus ventral sucker. Ovary lobed, submedian, pretesticular. pretesticular. Vitellarium follicular; follicles in two Seminal receptacle canalicular. Uterus pretesticular. lateral fields extending from posterior extremity to Vitellarium follicular; follicles in two lateral fields about halfway between intestinal bifurcation and extending from posterior extremity to level of ovary ventral sucker or further anterior. Excretory vesicle Y- or pharynx. Excretory vesicle Y-shaped; stem long, or T-shaped; stem short or long, occasionally with blind sigmoid. Intestinal parasites of birds; Far East. Type branches. Intestinal parasites of birds and mammals; and only genus Liliatrema Gubanov, 1953. cosmopolitan. Type genus Cryptocotyle Lühe, 1899. Other genus: Scaphanocephalus Jägerskiöld, 1903. Genus Liliatrema Gubanov, 1953

Subfamily Euryhelminthinae Morosov, 1952 Diagnosis: With characters of family. Type species L. skrjabini Gubanov, 1953. Syn.: Euryhelminae Morosov, 1952. The four subfamilies differ from the 12 (without the Diagnosis [modified from subfamily diagnosis of Pachytrematinae) earlier established opisthorchiid Yamaguti,(1971) and generic diagnosis of Pearson subfamilies (see: Scholz, 2008) most clearly in the (2008)]: Body rectangular to pentangular, or ovoid, following features: the Liliatrematinae, by the presence

© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–19 16 S. SOKOLOV ET AL. Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlaa093/5902681 by Federal Scientific Center of the East Asia user on 14 September 2020 of a specialized oral sucker, the uroproct and the genital dissolved or reduced to the status of a tribe or subtribe. sac without gonotyl; the Apophalinae, by the presence The current phylogenetic data and previously of a median permanent ventrogenital sac with a well- published materials (e.g. Thaenkham et al., 2012; developed ventral sucker and two gonotyls, which arise Tatonova et al., 2020) cast doubt on the monophyly of dorsally close against the ventral sucker overhanging the subfamily Opisthorchiinae. Thus, this subfamily it ventrally; the Cryptocotylinae, by the presence of a needs revision. median permanent ventrogenital sac with an anterior pocket and a rudimentary ventral sucker located on the large protractile bulge, but without gonotyl; ACKNOWLEDGEMENTS the Euryhelminthinae, by the presence of a median permanent ventrogenital sac with a well-developed This study was partly funded by the IEE RAS ventral sucker and an elliptical or reniform, largely Government basic research programme (project parenchymatose gonotyl. In this updated concept, the no. 0109-2018-0075). The authors declare that they family Opisthorchiidae has lost its former essential have no conflict of interest. difference with respect to Heterophyidae, which was the presence of the genital or the ventrogenital sac [compare with Bray (2008)]. The opistorchiids with an REFERENCES unspecialized (in particular, unarmed) oral sucker and the median genital or ventrogenital sac – apophallines, Andres MJ, Pulis EE, Cribb TH, Overstreet RM. 2014. cryptocotylines and euryhelminthines – differ from Erection of the haploporid genus Litosaccus n.g. and its the heterophyids with the same features in the phylogenetic relationship within the Haploporidae Nicoll, morphology of the ventral sucker or gonotyl (if any). 1914. Systematic Parasitogy 89: 185–194. Araki J, Machida M. 1990. Metacercaria of Liliatrema The liliatrematines possess a combination of features, sobolevi from greenling Hexagrammos agrammus. Japanese which does not occur among other opisthorchioids, Journal of Parasitology 39: 631–633. namely, the presence of a uroproct and a funnel- Atopkin DM, Besprozvannykh VV, Ha ND, Nguyen HV, shaped oral sucker with a penta- or heptagonal distal Khamatova AY, Vainutis KS. 2019. Morphometric and end, whose dorsal edge is covered with a hood-like molecular analyses of Carassotrema koreanum Park 1938 tegumental fold. At the same time, the differences and Elonginurus mugilus Lu 1995 (: Haploporidae) between the Opisthorchiidae and the Cryptogonimidae, Srivastava, 1937 from the Russian Far East and Vietnam. previously identified by Bray (2008), are still valid. Parasitology Research 118: 2129–2137. The phylogenetically based taxonomy of the Belden LK, Peterman WE, Smith SA, Brooks LR, Opisthorchioidea is still being developed. The current Benfield EF, Black WP, Yang Z, Wojdak JM. 2012. taxonomic model of this superfamily (see: Bray, 2008) Metagonimoides oregonensis (Heterophyidae: Digenea) is imperfect because of the polyphyly of the family infection in Pleurocerid snails and Desmognathus Heterophyidae, which was revealed repeatedly by the quadramaculatus salamander larvae in Southern nuclear DNA sequence analyses (Olson et al., 2003; Appalachian streams. The Journal of Parasitology 98: Sato et al., 2010; Thaenkham et al., 2011, 2012; Fraija- 760–767. Fernández et al., 2015; Le et al., 2017; Kuzmina et al., Belogurov OI, Leonov VA, Zueva LS. 1968. Helminthofauna 2018; Pantoja et al., 2018; Hernández-Orts et al., 2019; of fish-eating birds (gulls and guillemots) from the coast Kohl et al., 2019; Tatonova & Besprozvannykh, 2019; of the Sea of Okhotsk. In: Skrjabin KI, Mamaev YL, eds. this study). The present study and that of Tatonova & Helminths of of the Pacific Ocean. Moscow: Nauka, Besprozvannykh (2019) are the first attempts to resolve 105–124 [in Russian]. this problem. However, a discussion of the concrete Besprozvannykh VV, Tatonova YV, Shumenko PG. 2019. steps towards a further revision of the Heterophyidae Life cycle, morphology of developmental stages of Metorchis is currently impossible, because out of the several ussuriensis sp. nov. (Trematoda: Opisthorchiidae), and phylogenetic relationships with other opisthorchiids. Journal early-branching groups of these trematodes, only one of Zoological Systematics and Evolutionary Research 57: is statistically supported in different approaches of our 24–40. phylogenetic analysis (Figs 4A, 5A). Blair D, Barker SC. 1993. Affinities of the Gyliauchenidae: The absence of molecular data for 10 (without utility of the 18S rRNA gene for phylogenetic inference in the Pachytrematinae) opisthorchiid subfamilies the Digenea (Platyhelminthes). International Journal for (see: Scholz 2008), a labile position of Liliatrema, Parasitology 23: 527–532. Apophallus and Euryhelmis on 28S rDNA and Blasco-Costa, Balbuena JA, Kostadinova AA, Olson PD. 18S+28S rDNA-based trees, indicate that the proposed 2009. Interrelationships of the Haploporinae (Digenea: concept of the Opisthorchiidae is preliminary. In Haploporidae): a molecular test of the taxonomic framework future, the subfamilies Apophallinae, Cryptocotylidae based on morphology. Parasitology International 58: Euryhelminthinae and Liliatrematidae might yet be 263–269.

© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–19 SYSTEMATIC POSITION OF LILIATREMATIDAE 17 Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlaa093/5902681 by Federal Scientific Center of the East Asia user on 14 September 2020

Bray RA. 2008. Supertamily Opisthorchioidea Looss, 1899. In: Guindon S, Gascuel O. 2003. A simple, fast, and accurate Bray RA, Gibson DI, Jones A, eds. Keys to the Trematoda, Vol. algorithm to estimate large phylogenies by maximum 3. Wallingford: CABI International, 7–8. likelihood. Systematic Biology 52: 696–704. Bray RA, Cribb TH. 2012. Reorganisation of the superfamily Hernández-Orts JS, Georgieva S, Landete DN, Scholz T. Lepocreadioidea Odhner, 1905 based on an inferred 2019. Heterophyid trematodes (Digenea) from penguins: molecular phylogeny. Systematic Parasitology 83: 169–177. A new species of Ascocotyle Looss, 1899, first description of Bray RA, Webster BL, Bartoli P, Littlewood DTJ. 2005. metacercaria of Ascocotyle (A.) patagoniensis Hernández- A molecular phylogenetic study of the Acanthocolpidae Orts, Montero, Crespo, García, Raga and Aznar, 2012, and (Digenea). Acta Parasitologica 50: 281–291. first molecular data. International Journal for Parasitology: Bray RA, Waeschenbach A, Cribb TH, Weedall GD, Parasites and Wildlife 8: 94–105. Dyal P, Littlewood DTJ. 2009. The phylogeny of the Hostettler R, Cutmore SC, Cribb TH. 2018. Two new species Lepocreadioidea (Platyhelminthes, Digenea) inferred of Haplorchoides Chen, 1949 (Digenea: Heterophyidae) from nuclear and mitochondrial genes: implications for infecting an Australian siluriform fish, Neoarius graeffei their systematics and evolution. Acta Parasitologica 54: Kner & Steindachner. Systematic Parasitology 95: 201–211. 310–329. Huelsenbeck JP, Ronquist F, Nielsen R, Bollback JP. Bray RA, Foster GN, Waeschenbach A, Littlewood DTJ. 2001. Bayesian inference of phylogeny and its impact on 2012. The discovery of progenetic Allocreadium neotenicum evolutionary biology. Science 294: 2310–2314. Peters, 1957 (Digenea: Allocreadiidae) in water beetles Huston DC, Cutmore SC, Cribb TH. 2018. Molecular (Coleoptera: Dytiscidae) in Great Britain. Zootaxa 3577: systematics of the digenean community parasitising the 58–70. cerithiid gastropod Clypeomorus batillariaeformis Habe & Briscoe AG, Bray RA, Brabec J, Littlewood DT. 2016. Kusage on the Great Barrier Reef. Parasitology International The mitochondrial genome and ribosomal operon of 67: 722–735. Brachycladium goliath (Digenea: Brachycladiidae) recovered Jayawardena UA, Tkach VV, Navaratne AN, from a stranded minke whale. Parasitology International 65: Amerasinghe PH, Rajakaruna RS. 2013. Malformations 271–275. and mortality in the Asian Common Toad induced by Ciurea J. 1924. Heterophyidés de la faune parasitaire de exposure to pleurolophocercous cercariae (Trematoda: Roumanie. Parasitology 16: 1–21 [in French]. Cryptogonimidae). Parasitology International 62: 246–252. Ciurea J. 1933. Les vers parasites de l’homme, des mammiferes Kmentová N, Bray RA, Koblmüller S, Artois T, et des oiseaux provenant des poissons du Danube et de la De Keyzer ELR, Gelnar M, Vanhove MPM, Georgieva S. Mer Noire. Archives Roumaines de Pathologie Expérimentale 2020. Uncharted digenean diversity in Lake Tanganyika: et de Microbiologie 6: 5–134 [in French]. cryptogonimids (Digenea: Cryptogonimidae) infecting Cribb TH, Bray RA, Littlewood DTJ, Pichelin S, endemic lates perches (Actinopterygii: Latidae). Parasites & Herniou EA. 2001. Relationships of the Digenea – evidence Vectors 13: 221. from molecules and morphology. In: Littlewood DTJ, Bray RA, Kohl ZF, Calhoun DM, Elmer F, Peachey RBJ, Leslie KL, eds. Interrelationships of the Platyhelminthes. London and Tkach V, Kinsella JM, Johnson PTJ. 2019. Black-spot New York: Taylor & Francis, 186–193. syndrome in Caribbean linked to trematode parasite Dao THT, Nguyen TGT, Gabriel S, Bui KL, Dorny P, infection (Scaphanocephalus expansus). Coral Reefs 38: Le TH. 2017. Updated molecular phylogenetic data for 917–930. Opisthorchis spp. (Trematoda: Opisthorchioidea) from ducks Kostadinova A, Gibson DI. 2005. Family Liliatrematidae in Vietnam. Parasites & Vectors 10: 575. Gubanov, 1953. In: Jones A, Bray RA, Gibson DI, eds. Keys Darriba D, Taboada GL, Doallo R, Posada D. 2012. to the Trematoda, Vol. 2. Wallingford: CABI International, jModelTest 2: more models, new heuristics and parallel 679–682. computing. Nature Methods 9: 772. Kumar S, Stecher G, Tamura K. 2016. MEGA7: molecular Fraija-Fernández N, Olson PD, Crespo EA, Raga JA, evolutionary genetics analysis v.7.0. Molecular Biology and Aznar FJ, Fernández M. 2015. Independent host switching Evolution 33: 1870–1874. events by digenean parasites of cetaceans inferred from Kuzmina TA, Tkach VV, Spraker TR, Lyons ET, Kudlai O. ribosomal DNA. International Journal for Parasitology 45: 2018. Digeneans of northern fur seals Callorhinus ursinus 167–173. (Pinnipedia: Otariidae) from five subpopulations on St. Paul Frolov EV. 2010. Recording of metacercaria Liliatrema Island, Alaska. Parasitology Research 117: 1079–1086. sobolevi mtc. Gubanov, 1953 from greenlings Hexagrammos Kvach Y, Bryjová A, Sasal P, Winkler HM. 2018. The octogrammus and H. stelleri at south-eastern Sakhalin. taxonomic and phylogenetic status of digeneans from the Izvestiya of the Pacific Research and Fisheries Centre genus Timoniella (Digenea: Cryptogonimidae) in the Black (TINRO-Centre), Vladivostok 162: 338–341 [in Russian]. and Baltic seas. Journal of Helminthology 92: 596–603. Gubanov NM. 1953. A new trematode subfamily Le TH, Nguyen KT, Nguyen NTB, Doan HTT, Dung DT, Liliatrematinae nov. subfam. from marine fish-eating birds. Blair D. 2017. The ribosomal transcription units of In: Petrov AM, ed. Raboty po Gel’mintologii k 75-letiyu Haplorchis pumilio and H. taichui and the use of 28S Akademika K.I. Skrjabina. Moscow: Izdatel’stvo ANSSSR, rDNA sequences for phylogenetic identification of common 176–183 [in Russian]. heterophyids in Vietnam. Parasites & Vectors 10: 17.

© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–19 18 S. SOKOLOV ET AL. Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlaa093/5902681 by Federal Scientific Center of the East Asia user on 14 September 2020

Leonov VA, Belogurov OI, Shagvaleeva NM, representative Japanese populations. Parasitology Research Bondarenko SK. 1965. On the trematode fauna of fish- 115: 1123–1130. eating birds in Kamchatka. In: Leonov VA, Mamaev YL, Posada D. 2003. Using MODELTEST and PAUP* to select Oshmarin PG, eds. Paraziticheskie chervi domashnikh i a model of nucleotide substitution. Current Protocols in dikikh zivotnykh. Vladivostok: Akademiya Nauk SSSR, 130– Bioinformatics 6: 6.5.1–6.5.14. 158 [in Russian]. Price EW. 1940. A review of the trematode superfamily Literák I, Heneberg P, Sitko J, Wetzel EJ, Opisthorchioidea. Proceedings of the Helminthological Cardenas Callirgos JM, Čapek M, Valle Basto D, Society of Washington 7: 1–13. Papoušek I. 2013. Eye trematode infection in small Qiu YY, Gao Y, Li Y, Ma XX, Lv QB, Hu Y, Qiu HY, passerines in Peru caused by Philophthalmus lucipetus, Chang QC, Wang CR. 2020. Comparative analyses of an agent with a zoonotic potential spread by an invasive complete ribosomal DNA sequences of Clonorchis sinensis freshwater snail. Parasitology International 62: 390–396. and Metorchis orientalis: IGS sequences may provide a novel Littlewood DTJ, Olson PD. 2001. Small subunit rDNA and genetic marker for intraspecific variation. Infection, Genetics the Platyhelminthes: signal, noise, conflict and compromise. and Evolution 78: 104125. In: Littlewood DTJ, Bray RA, eds. Interrelationships of Sándor D, Molnár K, Gibson DI, Székely C, Majoros G, Platyhelminthes. London and New York: Taylor & Francis, Cech G. 2017. An investigation of the host-specificity 262–278. of metacercariae of species of Apophallus (Digenea: Lühe M. 1909. Parasitische Plattwürmer. I. Trematodes. In: Heterophyidae) in freshwater fishes using morphological, Brauer A, ed. Die Süsswasserfauna Deutschlands, Vol.17. experimental and molecular methods. Parasitology Research Jena: Verlag von Gustav Fischer, 1–217 [in German]. 116: 3065–3076. Machida M. 1966. Helminths collected from a pelagic shag. Sato H, Suzuki K. 2006. Gastrointestinal helminthes of feral Bulletin of the National Science Museum, Tokyo 9: 447–451. raccoons (Procyon lotor) in Wakayama Prefecture, Japan. Mehra HR. 1963. On a new family Liliatrematidae of Journal of Veterinary Medical Science 68: 311–318. the superfamily Echinostomatoidea Faust, 1929 with a Sato H, Ihara S, Inaba O, Une Y. 2010. Identification of discussion on its taxonomic position and a key to the families Euryhelmis costaricensis metacercariae in the skin of Tohoku of superfamily Echinostomatoidea. Proceedings of the hynobiid salamanders (Hynobius lichenatus), northeastern National Academy of Sciences, India. Section B 38: 240–244. Honshu, Japan. Journal of Wildlife Diseases 46: 832–842. Morozov FN. 1952. Superfamily Heterophyoidea Faust, 1929. Scholz T. 2008. Family Opisthorchiidae Looss, 1899. In: In: Skrjabin KI, ed. Trematodes of animals and man, Vol. 6. Bray RA, Gibson DI, Jones A, eds. Keys to the Trematoda, Vol. Moscow: Izdatel’stvo ANSSSR, 153–601 [in Russian]. 3. Wallingford: CABI International, 9–49. Ohbayashi M, Konno T. 1966. The metacercaria of Liliatrema Shumenko PG, Tatonova YV, Besprozvannykh VV. 2017. skrjabini Gubanov, 1953 from Sebastes schlegeli Hilgendorf. Metagonimus suifunensis sp. n. (Trematoda: Heterophyidae) Japanese Journal of Parasitology 15: 511–515 [in Japanese]. from the Russian Southern Far East: Morphology, life cycle, Ohbayashi M, Araki J. 1974. Metacercaria of Liliatrema and molecular data. Parasitology International 66: 982–991. sobolevi, Gubanov, 1953, from Hexagrammos otakii Jordan et Skorobrekhova EM. 2009. Encapsulation of the metacercariae Starks. Japanese Journal of Veterinary Research 22: 47–48. of two species of trematodes in the skin and musculature of Olson PD, Cribb TH, Tkach VV, Bray RA, Littlewood DT. the stone cockscomb Alectrias alectrolophus (Pallas, 1814). 2003. Phylogeny and classification of the Digenea Russian Journal of Marine Biology 35: 178–182. (Platyhelminthes: Trematoda). International Journal for Skrjabin KI, Koval VP. 1966. Allocreadiata, Allocreadiodea, Parasitology 33: 733–755. Nicoll, 1934. In Skrjabin KI, ed. Trematodes of animals and Pantoja CS, Hernández-Mena DI, de León GP, Luque JL. man, Vol. 22. Moscow: Izdatel’stvo ANSSSR, 177–456 [in 2018. Phylogenetic position of Pseudosellacotyla lutzi Russian]. (Freitas, 1941) (Digenea: Cryptogonimidae), a parasite of Sokolov S, Voropaeva E, Atopkin D. 2020. A new species Hoplias malabaricus (Bloch) in South America, through 28S of deropristid trematode from the sterlet Acipenser ruthenus rDNA sequences, and new observations of the ultrastructure (Actinopterygii: Acipenseridae) and revision of superfamily of their tegument. The Journal of parasitology 104: 530–538. affiliation of the family Deropristidae. Zoological Journal of Pearson JC. 2008. Family Heterophyidae Leiper, 1909. In: the Linnean Society zlaa015. Doi:10.1093/zoolinnean/zlaa015. Bray RA, Gibson DI, Jones A, eds. Key to the Trematoda, Vol. Tatonova YV, Besprozvannykh VV. 2019. Description of a 3. Wallingford: CABI International, 113–141. new species, Cryptocotyle lata sp. nov., and discussion of the Pérez-Ponce de León G, Hernández-Mena DI. 2019. phylogenetic relationships in Opisthorchioidea. Parasitology Testing the higher-level phylogenetic classification of Digenea International 72: 101939. (Platyhelminthes, Trematoda) based on nuclear rDNA Tatonova YV, Shumenko PG, Besprozvannykh VV. 2018. sequences before entering the age of the ‘next-generation’ Description of Metagonimus pusillus sp. nov. (Trematoda: Tree of Life. Journal of Helminthology 93: 260–276. Heterophyidae): phylogenetic relationships within the Pornruseetairatn S, Kino H, Shimazu T, Nawa Y, Scholz T, genus. Journal of Helminthology 92: 703–712. Ruangsittichai J, Saralamba NT, Thaenkham U. Tatonova YV, Besprozvannykh VV, Katugina LO, 2016. A molecular phylogeny of Asian species of the genus Solodovnik DA, Nguyen HM. 2020. Morphological Metagonimus (Digenea) – small intestinal flukes–based on and molecular data for highly pathogenic avian parasite

© 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–19 SYSTEMATIC POSITION OF LILIATREMATIDAE 19 Downloaded from https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlaa093/5902681 by Federal Scientific Center of the East Asia user on 14 September 2020

Erschoviorchis anuiensis sp. n. and phylogenetic relationships Tkach VV, Littlewood DT, Olson PD, Kinsella JM, within the Opisthorchiidae (Trematoda). Parasitology Swiderski Z. 2003. Molecular phylogenetic analysis of International 75: 102055. the Microphalloidea Ward, 1901 (Trematoda: Digenea). Thaenkham U, Dekumyoy P, Komalamisra C, Sato M, Systematic Parasitology 56: 1–15. Dung do T, Waikagul J. 2010. Systematics of the subfamily Truett GE. 2006. Preparation of genomic DNA from Haplorchiinae (Trematoda: Heterphyidae), based on nuclear tissues. In: Truett GE, Kieleczawa J, eds. The DNA book: ribosomal DNA genes and ITS2 region. Parasitology protocols and procedures for the modern molecular biology International 59: 460–465. laboratory. Sudbury: Jones and Bartlett Publisher, 33–46. Thaenkham U, Nawa Y, Blair D, Pakdee W. 2011. Wakabayashi S. 1997. Heavy infection of metacercariae of Confirmation of the paraphyletic relationship between Liliatrema skjabini (Digenea: Cathaemasiidae) in Jacopever families Opisthorchiidae and Heterophyidae using small rockfish Sebastes schlegeli. Bulletin Toyama Pretectural and large subunit ribosomal DNA sequences. Parasitology Fisheries Research Institute 9: 41–44 [in Japanese]. International 60: 521–523. Yamaguti S. 1958. Systema helminthum, Vol. I. New York & Thaenkham U, Blair D, Nawa Y, Waikagul J. 2012. Families London: Interscience Publishers. Opisthorchiidae and Heterophyidae: are they distinct? Yamaguti S. 1971. Synopsis of digenetic trematodes of Parasitology International 61: 90–93. vertebrates, Vol. 1. Tokyo: Keigaku Publishing Company. Tkach V, Pawlowski J, Mariaux J. 2000. Phylogenetic Zhukov EV. 1960. Endoparasitic worms of the fishes in analysis of the suborder plagiorchiata (Platyhelminthes, the Sea of Japan and South-Kuril shallow-waters. Trudy Digenea) based on partial lsrDNA sequences. International Zoologicheskogo Instituta Akademii Nauk SSSR 28: 3–146 Journal for Parasitology 30: 83–93. [in Russian].

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