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Sokolov, Sergey, Frolov, Evgeniy, Novokreshchennykh, Semen & Atopkin, Dmitry, 2021, An opisthorchiid concept of the genus Liliatrema (Trematoda: Plagiorchiida: Opisthorchioidea): an unexpected systematic position, Zoological Journal of the Linnean Society 192 (1), pp. 24-24 : 24-

Site of infection: Intestines.

Specimens deposited: Voucher specimen, IPEE RAS 1274.

Description (based on one specimen): Cirrus-sac absent. Seminal vesicle, pars prostatica with field of prostatic cells, and ejaculatory duct lying free in parenchyma. Seminal vesicle large, 298 in length, bipartite, with relatively short proximal portion and long distal portion, terminating in small sphincter, running dextrally to ventral sucker. Pars prostatica vesicular, surrounded by field of prostatic cells. Ejaculatory duct short, muscular, connecting with genital atrium. Genital atrium short, opening dextrosubmedially at base of genital sac. Genital sac with lip-like posterior wall overlapping anterior part of recessed ventral sucker; median.

PHYLOGENETIC DATA

The 28S-based maximum likelihood (ML) and Bayesian (BI) phylogenetic trees ( Fig. 4A, B ) demonstrate that Liliatrema falls within a large wellsupported clade, which also includes opisthorchiine genera Amphimerus Barker, 1911 , Clonorchis Loos, 1907 , Erschoviorchis Skrjabin, 1945 and Opisthorchis Blanchard, 1895 , a metorchiine genus Metorchis Looss, 1899 and the opisthorchiid (sensu Tatonova & Besprozvannykh, 2019) genus Apophallus Lühe, 1909 . Within this clade, Liliatrema appears as a sister-taxon to the poorly supported Apophallus + ( Opisthorchiinae + Metorchiinae) group on the BI tree ( Fig. 4B ), but forms a poorly supported group with Apophallus in the ML tree. The latter group occupies a sister-position in relation to the clade of traditional opisthorchiid genera ( Fig. 4 А). The Opisthorchiinae + Metorchiinae clade shows a distinct monophyly. However, the subfamily Opisthorchiinae , which is represented in our study by the genera Amphimerus , Clonorchis , Erschoviorchis and Opisthorchis , appears to be paraphyletic ( Fig. 4 А, B). Another opisthorchiid (sensu Tatonova & Besprozvannykh, 2019) genus, Cryptocotyle Lühe, 1899 , reliably clustered with the heterophyid genus Scaphanocephalus Jägerskiöld, 1903 . The phylogenetic position of the Cryptocotyle + Scaphanocephalus group is labile, depending on the phylogenetic algorithm used. This group appears as a sister-group to the opisthorchiid (sensu Tatonova & Besprozvannykh, 2019) genus Euryhelmis Poche, 1926 , with poor support on the BI tree ( Fig. 4B ). In turn, the Euryhelmis + ( Cryptocotyle + Scaphanocephalus ) and the Liliatrema + [ Apophallus + ( Opisthorchiinae + Metorchiinae)] clades are sister-groups. On the ML tree, the Cryptocotyle + Scaphanocephalus group appears as a sister-taxon to the poorly supported Euryhelmis + [( Liliatrema + Apophallus ) + ( Opisthorchiinae + Metorchiinae)] clade ( Fig. 4A ). The heterophyid genus Metagonimoides Price, 1931 has a basal position to all the trematodes mentioned above, but it is supported only in the ML tree ( Fig. 4A, B ). All the other heterophyids are distributed across three early-branching groups, but only one of these groups, the one containing the genera Galactosomum Looss, 1899 , Haplorchis Looss, 1899 , Haplorchoides Chen, 1949 , Metagonimus Katsurada, 1912 , Procerovum Onji & Nishio, 1916 and Stellantchasmus Onji & Nishio , has statistically significant supports on both ML and BI trees. Species of the Cryptogonimidae form a distinct clade, which is basal to the group including traditional opisthorchiids and the genera Apophallus , Cryptocotyle , Euryhelmis , Liliatrema and Scaphanocephalus ( Fig. 4A ).

Phylogenetic ML and BI analyses, based on concatenated complete 18S and partial 28S rRNA gene sequences, showed that Liliatrema is closely related to the Cryptocotyle + Euryhelmis group within a common highly supported clade, which also contains monophyletic group of the traditional opisthorchiid genera Clonorchis , Opisthorchis , Metorchis and Amphimerus ( Fig. 5A, B ). The Cryptocotyle + Euryhelmis group has high support on BI phylogenetic tree and moderate support on ML tree. The Opisthorchiinae appears as a paraphyletic group. Heterophyid species form two early-branching groups, which are united into a weakly supported clade. However, only one of these groups, the one that contains the genera Galactosomum , Haplorchis , Metagonimus , Procerovum and Stellantchasmus , is highly supported. The phylogenetic position of the Cryptogonimidae corresponds to that on the 28S-based trees ( Fig. 4A ).

Two variants of an opisthorchiid-specific 30-bp repeat within 361-bp ribosomal ITS1 were detected for L. skrjabini in positions 67–96 bp and 154–183 bp, respectively. The first repeat sequence variant is characterized by a unique nucleotide composition, containing a CCC triplet at positions 8–10.

Figures

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.

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.