applyparastyle “fig//caption/p[1]” parastyle “FigCapt” 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 genus Liliatrema (Trematoda: Plagiorchiida: 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 species. 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, Cryptocotyle 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 Opisthorchiidae. 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 fish other authors disagreed with this point of view (see: as second intermediate hosts and piscivorous birds 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 sucker 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 © 2020 The Linnean Society of London, Zoological Journal of the Linnean Society, 2020, XX, 1–19 1 2 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 (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). Allocreadiidae (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 ©