Introduction

Adult orientocreadiid trematodes typically inhabit the intestine of freshwater Eurasian and African siluriform and perciform fishes, but some species parasitise Indian terrestrial reptiles (Beverley-Burton 1962; Yamaguti 1971; Hafeezullah 1989). The systematic position of this group of parasites has repeatedly been discussed in the literature (e.g. Tubangui 1931; Yamaguti 1958; Skrjabin and Koval 1963; Fischthal and Kuntz 1963; Sirikantayakul 1985). According to the current point of view (Jones and Bray 2008), these trematodes belong to the separate family Orientocreadiidae Yamaguti 1958 within the superfamily Plagiorchioidea. This opinion has developed based on Fischthal and Kuntz (1963) taxonomic analysis of adult orientocreadiids and data on the morphology of Оrientocreadium batrachoides Tubangui, 1931 and Оrientocreadium pseudobagri Yamaguti, 1934 cercariae (Tang and Lin 1973; Besprozvannykh 1984). According to Jones and Bray (2008), the Orientocreadiidae is a monogeneric family with the following list of invalidated generic taxa that are congeneric with its type-genus—Оrientocreadium Tabangui, 1931, Ganada Chatterji, 1933, Neoganada Dayal, 1938, Nizamia Dayal, 1938, Ganadotrema Dayal, 1949, Macrotrema Gupta, 1951 nec Regan, 1912 and Paratormopsolus Bychowsky et Dubinina 1954. A total of 28 nominal species of orientocreadiids have been described (Yamaguti 1971; Agrawal and Sharma 1990; Shimazu 1990; Kim and Rim 1995; Besprozvannykh et al. 2009; Nigam et al. 2015); however, the validity of many of them is questionable (Hafeezullah 1989). Sequences of 28 rDNA have been used successfully as a data source for phylogenetic reconstruction within the superfamily Plagiorchioidea (Tkach et al. 1999, 2000a, b, 2001a, b; Pérez-Ponce de León et al. 2011; Hernández-Mena et al. 2016). In this paper, we investigate the phylogenetic position of the Orientocreadiidae inferred from the same fragment of DNA from one of the representatives of the genus Orientocreadium s. str.–О. pseudobagri.

Materials and methods

Specimens of О. pseudobagri (Fig. 1) were recovered from the intestine of Perccottus glenii (Dybowski, 1877) (Actinopterygii, Odontobutidae), caught in July 2010 in the water body with the working name “Ozero 1”, Primorsky Kray, Russia (Sokolov 2013). Trematodes were fixed in 70% ethanol and stained with acetocarmine. Some specimens were fixed in 96% ethanol for further molecular analysis. Trematode species were identified with the aid of the publications of Yamaguti (1934), Shimazu (1990, 2014), Kim and Rim (1995), Besprozvannykh et al. (2009) and Shimazu et al. (2011).

Fig. 1
figure 1

Orientocreadium pseudobagri from Perccottus glenii, Primorsky Kray, Russia. Scale bar 0.4 mm

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In order to obtain 28 rDNA sequence, total DNA was isolated with a ZymoBead Genomic DNA Kit (http://www.zymoresearch.com). Only single trematode specimens were used for each DNA extraction. The DNA fragment of about 1200 bp localised at the 5′ end of 28 rDNA was amplified using the BIO-RAD C1000 Thermal Cycler. PCR were performed in a total volume of 20 μl (11.5 μl) H20, 2.5 μl Taq buffer, 2 μl dNTP at concentration 10 pM, 0.5 μl of each primer at concentration 10 pM, 1 μl of Taq polymerase (“Syntol”) and 1 μl of DNA template.

Trematode-specific forward primer LSU-5 (5′-TAG GTC GAC CCG CTG AAY TTA AGC A-3′) and reverse primer 1500R (5′-GCT ATC CTG AGG GAA ACT TCG-3′) were used. Genbank numbers of sequences used in analysis are provided in the Table 1. Thermal cycle parameters were as follows: initial denaturation at 95 °C (3 min); 35 cycles of 20 s at 95 °C; 20 s at 56 °C; 120 s at 72 °C; 5 min at 72 °C for final extension. Amplicons were purified using Cleanup mini Purification Kit (Eurogene). All amplicons were sequenced directly using the equipment of the Research Park of Saint-Petersburg State University (Centre for Molecular and Cell Technologies). Sequences from both forward and reverse primers were assembled using Chromas Pro 1.7.4.

Table 1 List of species, incorporated into molecular analysis: systematic affiliation by Olson et al. (2003) with additions (Overstreet and Curran 2005; Choudhury et al. 2007; Bray 2008; Bray and Cribb 2012; Heneberg and Literák 2013; Kanarek et al. 2014; Besprozvannykh et al. 2015a; Shedko et al. 2015; Bray et al. 2016; Littlewood et al. 2015; Hernández-Mena et al. 2016; Martínez-Salazar et al. 2016)
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Obtained sequences were included in the general alignment (Table 1). In total, 133 sequences (in addition to the newly obtained one) were used for alignment. First, sequences were automatically aligned using Muscle algorithm (Edgar 2004), as implemented in SeaView 4.0 (Gouy et al. 2010), followed by manual alignment verification. The phylogenetic analysis was performed using the maximum likelihood method (ML) with GTR + G + I model. In total, about 1100 sites were selected for the analysis. The ML phylogenetic tree was obtained using RaxML program (Stamatakis 2006) at CIPRES Science Gateway (www.phylo.org) (Miller et al. 2010). The stability of clades was assessed using a non-parametric bootstrap with 1000 pseudoreplicates. All model parameters were estimated from the data. Bayesian inference analysis (BI) was performed using MrBayes 3.1.2, GTR model with gamma correction for inter-site rate variation (8 categories) and the covarion model. Trees were run as two separate chains (default heating parameters) for 15 million generations at which point they had ceased converging. The quality of chains was estimated using built-in MrBayes tools and additionally using Tracer 1.6 (Rambaut et al. 2014). Based on the estimates by Tracer, 50,000 generations were discarded for burn-in (relative burn-in parameter was switched off).

Results

The general topology of the trees constructed by ML and BI was almost coincided (Fig. 2). Incongruent branches are labelled with an asterisk (*). In most of the cases, the mismatches of branching in ML and BI are caused by settings of BI analysis (because all trees with tripartitions were excluded).

Fig. 2
figure 2

Bayesian tree of the Orientocreadiidae based on the analysis of 28S rDNA partial sequences. Nodal numbers are indicated: bootstrap value to the left from slash mark and Bayesian statistics to the right; only significant values are shown (above 80% for bootstrap value and 0.9 for BI). Sequence of Echinostoma revolutum is used as outgroup

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Phylograms from both ML and BI placed the Orientocreadiidae (=O. pseudobagri) within a major clade corresponding to the superfamily Plagiorchioidea (see Table 1) with the family Leptophallidae supported as the sister taxon (Fig. 2). In turn, the clade of Orientocreadiidae + Leptophallidae has a strongly supported sister relationship with the clade of the Alloglossidiidae. The monophyletic clade uniting orientocreadiids, leptophallids and alloglossidiids grouped into a large weakly supported clade containing members of the families Brachycoeliidae, Choanocotylidae, Glypthelminthidae, Auridistomidae and Macroderoididae. In all cases, the Auridistomidae and Macroderoididae appeared sister groups, as did the Brachycoeliidae and Choanocotylidae + Glypthelminthidae (with strong and weak support, respectively). BI analysis revealed that the group of Auridistomidae + Macroderoididae is aggregated with the Alloglossidiidae + (Orientocreadiidae + Leptophallidae) clade, while the Brachycoeliidae + (Choanocotylidae + Glypthelminthidae) clade occupied a sister position to all the former mentioned trematodes. In ML analysis, Auridistomidae + Macroderoididae appear as a weakly supported sister clade to that formed by the brachycoeliids, choanocotylids and glypthelminthids.

Other plagiorchioid trematodes analysed in the phylogenetic reconstruction are the Haematoloechidae, Omphalometridae, Plagiorchiidae, Telorchiidae, Cephalogonimidae, Reniferidae and the genera Choledocystus Pereira et Cuocolo, 1941, Infidum Travassos, 1916, and Rauschiella Babero, 1951 (as Plagiorchioidea incertae sedis by Hernández-Mena et al. (2016) and Martínez-Salazar et al. (2016)) are basal taxa to orientocreadiids. The families Haematoloechidae, Omphalometridae, Plagiorchiidae, Reniferidae and the clade of Choledocystus + Infidum are resolved inside it as well-supported monophyletic groups. In the same time, intergeneric relationships of the telorchiids and cephalogonimids were poorly resolved with the exception of node Cephalogonimus retusus (Dujardin, 1845)/Opisthioglyphe ranae (Frölich, 1791) in BI analysis. Position of the genus Rauschiella on phylograms that are produced by different methods is unstable.

Discussion

Our molecular data reveal a close phylogenetic relationship between orientocreadiids and both alloglossidiids and leptophallids, which justifies the placement of the Orientocreadiidae in the Plagiorchioidea. Previously, Hernández-Mena et al. (2016) showed molecular evidence of phylogenetic affinity between alloglossidiids and leptophallids. Analysis of the sequences obtained in the present study demonstrated a closer relationship between leptophallids with orientocreadiids than with the alloglossidiids. Dayal (1938) was the first to notice that orientocreadiids (at that time attributable to the genera Ganada, Neoganada and Nizamia) are morphologically close to leptophallids. An indisputable synapomorphy of the Orientocreadiidae + Leptophallidae clade is the presence of an external seminal vesicle. The external seminal vesicle in both families is unipartite, tubular or saccular, without associated gland cells (Tkach et al. 1999; Shimazu 2014). Within the Plagiorchioidea, this organ is characteristic only of representatives of the said clade (Bray 2008). In general, this structure is not unique to plagiorchioids and appears with varying frequency in other superfamilies of trematodes, in particular, in the Opecoeloidea and Lepocreadioidea. The most significant morphological difference between adult leptophallids and orientocreadiids is the presence of a canalicular seminal receptacle in the Leptophallidae. In orientocreadiids, there is a uterine seminal receptacle (Bray 2008).

Leptophallid and orientocreadiid trematodes are parasites of different groups of vertebrates. Adult leptophallids are parasites of the intestine or lungs of snakes (Tkach 2008) and orientocreadiids mainly parasitise the intestine of the ray-finned fishes (Actinopterygii). Only two species of the family have been described from terrestrial lizards (Hafeezullah 1989), one of which—Orientocreadium ottoi Agrawal, 1966—is considered by some authors as conspecific with О. batrachoides, a parasite of the catfishes (Pandey 1970; Hafeezullah 1989).

The first intermediate hosts of the trematode groups in question are pulmonate snails—Lymnaeidae for orientocreadiids (Tang and Lin 1973; Besprozvannykh 1984; Sirikantayakul 1985; Besprozvannykh et al. 2009), and Lymnaeidae or Planorbidae for leptophallids (Grabda-Kazubska 1963; Dobrovolʼski 1969). Xiphidiocercariae of orientocreadiids and leptophallids are similar in general morphology. These larvae have a relatively large bodies (about 0.3 mm in length), anterior organs with a stylet of an “open type” (the small bulb is not covered), and a relatively long prepharynxes (Tkach et al. 1999; Besprozvannykh et al. 2009). The excretory bladder in orientocreadiid and many leptophallid cercariae is Y-shaped, thick-walled, with the terminal mouths of the main collecting ducts (Tkach et al. 1999; Besprozvannykh et al. 2009). Only in representatives of the genus Macrodera Lühe, 1899 do the main collecting ducts open into the arms subterminally (Tkach et al. 1999). The protonephridial formula is 2[(3 + 3 + 3) + (3 + 3 + 3)] = 36 in all known cercariae in both families. Cercariae of leptophallids, however, have 4 or 8 pairs of non-differentiated penetration glands (Tkach et al. 1999), whereas only 3 or 5 pairs of penetration glands have been reported for orientocreadiid cercariae (Tang and Lin 1973; Besprozvannykh et al. 2009).

General topology of orientocreadiid cercarial sensillae demonstrates great similarities with other plagiorchioid trematodes including leptophallids (Besprozvannykh et al. 2009). The presence of numerous sensillae on the anterior end of O. pseudobagri cercariae (well expressed “C”-circles and groups of “St”), as well as AID row (equal to StD3 group in Besprozvannykh et al. 2009) and two S-circles (9S1 and 5S2 according to Besprozvannykh et al. 2009), is consistant with the plagiorchioid type of the chaetotaxy. Nevertheless, the main character, which approximates O. pseudobagri cercaria with other species of plagiorchioid trematodes, is the presence of 2 UD (“U” in Besprozvannykh et al. 2009) sensillae on tail tegument.

Miracidia of representatives of the genus Orientocreadium are very similar to those at other plagiorchioid species including leptophallids. They have the same epithelial formula (3 + 3) and related glandular apparatus—two large penetration glands situated immediately posterior to the terebratorium (Tang and Lin 1973). Unfortunately, nothing is known about organisation of the excretory system and germinative primordium in orietocreadiid miracidia. In addition, even less is known about sporocysts of leptophallids and orientocreadiids other that they have an elongated body with a thick wall. In all studied species, the birth pore is situated terminally (Dobrovolʼski 1969; Tang and Lin 1973; Besprozvannykh et al. 2009).

No morphological synapomorphy is apparent for the clade of Alloglossidiidae + (Orientocreadiidae + Leptophallidae). External phylogenetic connections of this group with other plagiorchioid trematodes cannot yet be adequately identified.

In general, only the Telorchiidae among the nine families of the Plagiorchioidea (represented in our study by more than one species) has not been demonstrated to be monophyletic. The Telorchiidae is represented here by members of two subfamilies—Telorchiinae (Telorchis assula (Dujardin, 1845)) and Opisthioglyphinae (Opisthioglyphe ranae (Frölich, 1791)) (see Font and Lotz 2008). The association of these taxa into one family is supported by data on the cercarial chaetotaxy (Grabda-Kazubska and Lis 1993) and the results of the molecular study by Tkach et al. (2000a). However, the molecular data of a number of subsequent authors testify to the paraphyly of the Telorchiidae (Olson et al. 2003; Bray et al. 2005; Pérez-Ponce de León et al. 2011; Martínez-Salazar et al. 2016).