Three members of Opisthomonorcheides Parukhin, 1966 (Digenea: Monorchiidae) from carangid fishes (Perciformes) from Indonesia, with a review of the genus

Three species of Opisthomonorcheides Parukhin, 1966 are reported for the first time from Indonesian waters: O. pampi (Wang, 1982) Liu, Peng, Gao, Fu, Wu, Lu, Gao & Xiao, 2010 and O. ovacutus (Mamaev, 1970) Machida, 2011 from Parastromateus niger (Bloch), and O. decapteri Parukhin, 1966 from Atule mate (Cuvier). Both O. pampi and O. ovacutus can now be considered widespread in the Indo-Pacific region, with earlier records of these species being from Fujian Province, China and Penang, Malaysia, respectively. We redescribe O. decapteri from one of its original hosts, Atule mate, off New Caledonia, and report this species from Jakarta Bay, Indonesia, extending its range throughout the Indian Ocean into the south-western Pacific. All three species possess a genital atrium that is long, sometimes very long, and a genital pore that is located in the forebody. This validates the interpretation that the original description was erroneous in reporting the genital pore in the hindbody, well posterior to the ventral sucker. These observations verify the synonymy of Retractomonorchis Madhavi, 1977 with Opisthomonorcheides. A major discrepancy between the species of Opisthomonorcheides is that some are described with the uterus entering the terminal organ laterally and some with it entering terminally; this feature needs further analysis. Based on the length of the genital atrium and the posterior extent of the vitellarium, the 27 species of Opisthomonorcheides considered valid can be divided into four groups. Among the 53 host records analysed, the families Carangidae (53% of records), Stromateidae (17%) and Serranidae (5.7%) are the most common; the reports are overwhelmingly from members of the Perciformes (91%), with further records in the Clupeiformes (5.7%), Gadiformes (1.9%) and Pleuronectiformes (1.9%). Two fish genera (Parastromateus Bleeker and Pampus Bonaparte) dominate the recorded hosts, with the black pomfret Parastromateus niger harbouring six species, the silver pomfret Pampus argenteus (Euphrasen) harbouring six, and the Chinese silver pomfret P. chinensis (Euphrasen) two. A host-parasite checklist is presented. We discuss the host-specificity of members of the genus, questioning some records such as that of O. decapteri in a deep-sea macrourid. We also comment on the morphological similarity, but phylogenetic distance, between the various Pomfret species, advancing the possibility that a series of host misidentifications has occurred. Sequences of the ITS2 rDNA gene generated for O. pampi and O. ovacutus are briefly discussed and molecular data are lodged in the GenBank database.

extending its range throughout the Indian Ocean into the south-western Pacific. All three species possess a genital atrium that is long, sometimes very long, and a genital pore that is located in the forebody. This validates the interpretation that the original description was erroneous in reporting the genital pore in the hindbody, well posterior to the ventral sucker. These observations verify the synonymy of Retractomonorchis Madhavi, 1977 with Opisthomonorcheides. A major discrepancy between the species of Opisthomonorcheides is that some are described with the uterus entering the terminal organ laterally and some with it entering terminally; this feature needs further analysis. Based on the length of the genital atrium and the posterior extent of the vitellarium, the 27 species of Opisthomonorcheides considered valid can be divided into four groups. Among the 53 host records analysed, the families Carangidae (53% of records), Stromateidae (17%) and Serranidae (5.7%) are the most common; the reports are overwhelmingly from members of the Perciformes (91%), with further records in the Clupeiformes (5.7%), Gadiformes (1.9%) and Pleuronectiformes (1.9%). Two fish genera

Introduction
Fish parasitological studies in Indonesian waters demonstrate the difficulty of an enormous biodiversity confronted by a meagre research effort and a lack of competent investigations. Although several new parasite species have been described in recent years (e.g. Palm, 2004;Bray & Palm, 2009;Yong et al., 2016), the fauna remains largely unknown ; it can be expected that many more species await description and many other known species from the Indo-Pacific region also occur off Indonesia (see Kuchta et al., 2009). Consequently, taxonomic treatments of nearly all parasite taxa in the region are needed.
During the First Educational Workshop on Fish Parasitology in Indonesia during 2013, Indonesian and international students and researchers investigated a wide range of fishes from Balinese waters. We herein report three species of Opisthomonorcheides Parukhin, 1966, a genus not yet recorded off Indonesia, from two carangid species. This genus was erected by Parukhin (1966) for a species reported from Decapterus sp. and Atule mate (Cuvier) in the Gulf of Tonkin, described with a genital pore in the hindbody well posterior to the ventral sucker. Madhavi (1977) later described a new genus, Retractomonorchis Madhavi, 1977(for R. delicatus Madhavi, 1977, from Pampus chinensis (Euphrasen) and P. argenteus (Euphrasen) off the Waltair Coast, India. She described the genus with a genital pore in the posterior forebody and a thin-walled genital atrium reaching into the hindbody. In some cases, the genital atrium was difficult to detect and she indicated that, if it had been missed by Parukhin (1966), then her worms were congeneric with his. Hafeezullah (1984) studied over 40 specimens of Opisthomonorcheides karwarensis Karyakarte & Yadav, 1976 and also found the genital atrium difficult to distinguish in some cases and, presuming Parukhin (1966) to be mistaken, synonymised Retractomonorchis with Opisthomonorcheides. In her review of the Monorchiiidae, Madhavi (2008) agreed with Hafeezullah's conclusions and also synonymised Longimonorchis Mamaev, 1970 with Opisthomonorcheides. She also considered Tritesticulum Bilqees, 1971 a synonym, but says that 'its description is inadequate and probably erroneous'. Taking Bilqees' (1971) description at face value, it does not resemble species of Opisthomonorcheides, but the worm is probably unrecognisable. Species of Opisthomonorcheides are clearly distinguished from species of the two other genera of the subfamily Opisthomonorcheidine Yamaguti, 1971 by the presence of a single testis rather than two testes (Madhavi, 2008). Machida (2011) described worms identified as belonging to the type-species of Opisthomonorcheides, O. decapteri Parukhin, 1966, from Rastrelliger kanagurta (Cuvier) off Palawan, the Philippines, with a long genital atrium and the genital pore in the posterior forebody. We have available some specimens which we consider O. decapteri, from one of its original hosts, Atule mate (no type-host was designated) originating from the Fish Market in Nouméa, New Caledonia, and Jakarta Bay, Indonesia. We include a description and illustration of these worms to show that the genital pore is in the forebody and the genital atrium is long, thus validating the presumptions of Madhavi (1977Madhavi ( , 2008 and Hafeezullah (1984) and agreeing with Machida (2011). The species O. pampi (Wang, 1982) Liu, Peng, Gao, Fu, Wu, Lu, Gao & Xiao, 2010, O. ovacutus (Mamaev, 1970 Machida, 2011 and O. decapteri are described for the first time from Indonesian waters.

Materials and methods
During the First Educational Workshop on Fish Parasitology in Indonesia, July 21st -August 2nd, 2013, at the Faculty of Veterinary Sciences, Denpasar, Bali, Indonesia, a single fresh specimen of Parastromateus niger (Bloch) was bought at Kedonganan fish market and transported on ice to the laboratory for study. Digeneans were collected live, immediately fixed in nearly boiling saline and stored in 70% ethanol. Whole-mounts were stained with Mayer's paracarmine, cleared in beechwood creosote and mounted in Canada balsam. Specimens of Atule mate were collected from Jakarta Bay on the 12th of January, 2012, deep frozen and transported to UNSOED University, Purwokerto, for subsequent study. A specimen of P. niger, originating from off Tegal, northern coast of Central Java, was collected in June, 2011 at the fish market in Cilacap, south coast of Central Java, and studied in the laboratory of UNSOED University, Purwokerto. Wholemounts of trematodes from these hosts were fixed and stored in 70% ethanol and later mounted in glycerine.
Measurements were made using a drawing tube attached to an Olympus BH-2 microscope, using a Digicad Plus digitising tablet and Carl Zeiss KS100 software adapted by Imaging Associates, and are given in micrometres. Measurements taken from worms from frozen hosts are not included in the tables. The following abbreviations are used: BMNH, the British Museum (Natural History) Collection at the Natural History Museum, London, UK; NBC, National Biodiversity Collection, Lipi-Cibinon, Bogor, Java, Indonesia; ZMB, Natural History Museum, Berlin, Germany; MNHN JNC, Muséum National d'Histoire Naturelle, Paris, France.
Genomic DNA of two species, O. pampi and O. ovacutus, was isolated and purified from individual specimens using a genomic DNA extraction kit (Peqlab Biotechnology GmbH, Erlangen, Germany) according to the manufacturer's instructions. The ITS2 rDNA region was amplified by using the forward primer 3S (5 0 -GGT ACC GGT GGA TCA CGT GGC TAG TG-3 0 ) (Morgan & Blair, 1995) and the reverse primer ITS2.2 (5 0 -CCT GGT TAG TTT CTT TTC CTC CGC-3 0 ) (Cribb et al., 1998). PCR-reactions (25 ll) included 2 ll of genomic DNA and 23 ll of Master-Mix (Peqlab Biotechnology GmbH, Erlangen, Germany) containing 4 mM MgCl 2 , 40 mM Tris-HCl (pH 8.55), 32 mM (NH 4 ) 2 SO 4 , 0.02% Tween 20 (ready mixed as Peqlab ''Reactionbuffer Y'', 2.5 ll) and 2.5 u Taq-Polymerase (0.5 ll), 0.4 mM dNTP (1.25 ll) and 16.75 ll water and 1 ll of each primer (10 pmol/ll). Amplification was performed in a thermocycler (Biozym Scientific GmbH, Germany) under the following conditions: an initial single cycle of 95°C denaturation for 3 min, 45°C annealing for 2 min, 72°C extension for 90 s, followed by 4 cycles of 95°C denaturation for 45 s, 50°C annealing for 45 s, 72°C extension for 90 s, followed by 30 cycles of 95°C denaturation for 20 s, 52°C annealing for 20 s, 72°C extension for 90 s, followed by a final 72°C extension for 5 min (Cutmore et al., 2013). Control samples without DNA were included in each PCR run. PCR products were examined on 1% agarose gels. A 100 bp ladder marker (Peqlab Biotechnology GmbH, Erlangen, Germany) was used to estimate the size of the PCR products. To identify the specimens, the PCR products were purified with Cycle-Pure Kit (Peqlab Biotechnology GmbH, Erlangen, Germany) and sequenced by Seqlab (Goettingen GmbH, Germany) using 3 ll of primer (of each 3S and ITS2.2) and 7 ll of the PCR product (* 250 ng/ll). Sequencher TM version 4.5 (GeneCodes Corp.) was used to assemble and edit contiguous sequences and the start and the end of the ITS2 region were determined by annotation through the ITS2 Database (Keller et al., 2009;Ankenbrand et al., 2015)  Opisthomonorcheides pampi (Wang, 1982)  Ovary clearly tripartite, although overall outline may be weakly tri-lobed or almost circular, in posterior half of hindbody, pre-testicular, distant from testis. Uterine seminal receptacle present. Mehlis' gland and Laurer's canal obscured by eggs. Uterus reaches from about mid-way between ventral sucker and ovary to posterior part of post-testicular region; distal extremity passes ventrally to cirrus-sac. Eggs numerous, small. Metraterm not differentiated, terminal part of uterus passes into anterior part of terminal organ through sphincter. Terminal organ lies alongside or ventral to cirrus-sac, of similar length to ejaculatory duct, lined with large spines which may appear to extend into base of genital atrium. Vitellarium follicular; follicles unite to form lobulate strings, in lateral fields reaching from about mid-way between ventral sucker and ovary to close to posterior extremity.
Testis single, oval, close to posterior end of body. Cirrus-sac claviform, reaches to about mid-point between ventral sucker and ovary. Internal seminal vesicle oval, in posterior third of cirrus-sac. Pars prostatica elongate, narrow, with distinct gland-cell ducts arcing around it. Ejaculatory duct wide, narrows gradually anteriorly, lined with long spines. Genital atrium distinct, narrow, just reaches into hindbody or only reaches dorsally to ventral sucker. Genital pore median, in posterior forebody.
Ovary clearly tripartite, although overall outline may be weakly tri-lobed or almost circular, in posterior half of hindbody, pre-testicular, close to testis. Uterine seminal receptacle present. Mehlis' gland obscured by eggs. Laurer's canal opens dorsally to ovary. Uterus reaches from about mid-way between ventral sucker and ovary to posterior part of posttesticular region; distal extremity passes ventrally to cirrus-sac. Eggs numerous, small. Metraterm not differentiated, terminal part of uterus passes into anterior part of terminal organ. Terminal organ lies alongside or ventral to cirrus-sac, of similar length to ejaculatory duct, lined with large spines which may extend into base of genital atrium. Vitellarium follicular; follicles unite to form lobulate strings in lateral fields, reaching from about mid-way between ventral sucker and ovary to close to posterior extremity.
Testis single, oval, fairly close to posterior extremity of body. Cirrus-sac claviform, reaches to about two thirds of distance from ventral sucker to ovary. Internal seminal vesicle oval, in posterior half of cirrus-sac. Pars prostatica elongate, narrow, with distinct gland-cell ducts arcing around it. Ejaculatory duct wide, lined with long spines. Genital atrium long, narrow or inflated, reaches well into hindbody. Genital pore median, in posterior forebody.
Ovary clearly tripartite, although overall outline may be weakly tri-lobed or almost circular, in midhindbody, pre-testicular, distant from testis. Uterine seminal receptacle observed posterior and anterior to ovary. Mehlis' gland and Laurer's canal obscured by eggs. Uterus reaches from about mid-way between ventral sucker and ovary to posterior part of posttesticular region; distal extremity passes ventrally to cirrus-sac. Eggs numerous, small. Metraterm not differentiated, terminal part of uterus passes into anterior part of terminal organ. Terminal organ lies alongside or ventral to cirrus-sac, of similar length to ejaculatory duct, lined with large spines. Vitellarium follicular, in lateral fields reaching from just posterior to mid-way between ventral sucker and ovary to anterior edge of testis.
Opisthomonorcheides decapteri Parukhin, 1966 See above for summary of reports.

Species
O. pampi (Wang, 1982) O. ovacutus (Mamaev, 1970) O  Mhaisen et al. (2013) noted that E. tauvina is not found in the Arabian Gulf and that this is probably a misidentification of E. coioides (Hamilton); their interpretation is accepted here.

Remarks
The list of reports of this species is given above. We have interpreted some of the new specimens from off  (Wang, 1982) O. ovacutus (Mamaev, 1970) O Bali as belonging to this species. The caeca were originally described as 'united' at the posterior extremity by Wang (1982). However, the terminations of the caeca are difficult to detect amongst the eggs in the new specimens, although, in a few cases, it is apparent that they end blindly. As this feature is difficult to detect in our specimens, we do not think this is a reliable feature to distinguish these forms. The illustration in the original description indicates that the uterus enters the terminal organ from the side.

Remarks
The ovary of this species is reported as being bilobed and the prepharynx and oesophagus as being of similar length. The metraterm is described and illustrated as entering the base of the terminal organ. These characteristics serve to distinguish this species from O. pampi.  Ahmad (1977).

Remarks
This species was pre-occupied and has been renamed four times (Pozdnyakov, 1981;Ahmad, 1985b;Ahmad & Gupta, 1985;Gupta & Puri, 1985). The name O. puriensis was erected by both Ahmad (1985b) and Ahmad & Gupta (1985). The point of entry of the uterus into the terminal organ is not described, but the illustration suggests that it enters the base.

Remarks
This is a distinctive species, with the testis close to the posterior extremity. The ovary is oval, the metraterm is described and illustrated as entering the base of the terminal organ and the oesophagus is much longer than the prepharynx.

Remarks
This species is distinctive in having an equatorial ventral sucker and an oesophagus much longer than the prepharynx. The metraterm is described and illustrated as entering the base of the terminal organ.

Remarks
This species was originally described with a bipartite seminal vesicle and the metraterm is described and illustrated as entering the base of the terminal organ (Gupta & Puri, 1985). Similarly, the seminal vesicle is described as bipartite and the uterus is described as entering the distal end of the terminal organ in the description of O. engraulisi (see Gupta & Singh, 1990). We considered O. engraulisi a junior synonym of O. simhai as it described from the same host, in the northern waters of the Indian Ocean, and we can detect no significant differentiating features.

Remarks
This species is distinctive in possessing a vitellarium that is interrupted at the level of the ovary, a testis that is at the posterior extremity, a long oesophagus and a short prepharynx. The ovary is described as spherical and the metraterm is described and illustrated as entering the base of the terminal organ.  (4).

Remarks
According to Hafeezullah (1984), Opisthomonorcheides indicus Karyakarte & Yadav, 1976, O. indicus Ahmad, 1977, O. indicus Gupta & Gupta, 1978, R. delicatus Madhavi, 1977  decapterus. It appears that this species is valid, being the oldest species with a pretesticular vitellarium and a short genital atrium. The terminal organ is not described or illustrated in the original description. We see no reason to disagree with the opinion of Hafeezullah (1984) who considered O. delicatus a synonym of O. karwarensis (under its original name Retractomonorchis delicatus). The uterus is described as entering the distal end of the terminal organ, which, judging from the illustration, likely means the base (Madhavi, 1977).
Similarly, we agree that O. nigeri is a synonym of O. karwarensis. Although Hafeezullah (1984)  to O. nigeri. The terminal organ was not described or illustrated by Gupta & Puri (1981).

Remarks
This species is unusual in the possession of an entire ovary and, more especially, in the vitellarium reaching to the level of the ventral sucker. The uterus is described and illustrated as entering the base of the terminal organ.

Remark
The list of reports of this species is given above. The uterus is described and illustrated as entering the anterior part of the side of the terminal organ.

Remarks
This species was reported to have a vitellarium that reaches the 'posterior tip of the testis', thus differing from O. ovacutus. The terminal organ was not described.

Remarks
The status of this species remains unclear. It is reported to possess an unlobed ovary and a vitellarium that reaches to mid-testicular level. The uterus is described as entering the base of the terminal organ.

Remarks
For this species, which was renamed by Gupta & Puri (1985), the posterior vitelline extent is described as 'either posterior 1st part or, hind end or some distance back of testis'. The uterus is described as 'opening into terminal organ near its posterior side'.

Remarks
This species has a vitellarium described as 'extending from a little posterior to posterior margin of acetabulum to hind end of testis'. The vitelline extent, therefore, does not coincide with that of O. ovacutus. The uterus is described as entering the base of the terminal organ.

Remarks
Madhavi (2011) used the combination O. overstreeti, while fully aware that it is a homonym of O.
overstreeti Ahmad 1985. The two names in question were erected in the same volume and part of the journal, but Retractomonorchis overstreeti was introduced on p. 19 and Opisthomonorcheides overstreeti on p. 250. According to the ICZN Article 24 (and see comment on http://iczn.org/content/there-suchthing-%E2%80%9Cpage-priority%E2%80%9D), priority relies on the actions of the first reviser, not page priority. As this name is a subsequent combination, the other species (O. overstreeti Ahmad, 1985) is here given priority (we consider the present study to be the first revision). The ovary is described as entire, and the worm is relatively squat, thus differing from O. ovacutus. The uterus is described as entering the base of the terminal organ.

Remark
The uterus is described as entering at the 'distal end' of the terminal organ. This is not clear in the illustration of Gupta & Puri (1985).

Remarks
In the original description, the ovary is defined as being unlobed, but it is indented in the illustration. The uterus is described as entering the 'distal' end of the terminal organ, meaning the base, judging from the illustration. Opisthomonorcheides stromatensis Ahmad & Gupta, 1985 28 emend.

Remark
The uterus is described as entering the base of the terminal organ.

Remarks
The ovary is described as 'unlobed' and drawing of this species shows it to be relatively squat. The uterus is described as entering the 'distal' end of the terminal organ.

Remarks
This species is drawn as rather squat and the testis is relatively tiny thus differing from O. ovacutus. The terminal organ is not described or illustrated.

Overview
In our view, the recognition of species in this genus is unusually problematic. It is unlikely that all nominal species are valid given the poor standard of many descriptions and the lack of critical comparison of the species at the time of description. However, we refrain from proposing sweeping synonymies because we think the issues are further confused by morphological uncertainty (especially with respect to the nature of the terminal genitalia) and host specificity (especially with respect to the infection of species of Pampus and Parastromateus but also with respect to the significance of infection of other groups of fishes might reasonably be considered distinct species. In addition, we note that we are unable to predict with any reliability whether there might be a rich complex of species of Opisthomonorcheides within individual fish species as has been shown for some other combinations of fish and trematodes (e.g. Miller & Cribb, 2007 Machida (2011). A number of the figures of terminal organs with a posterior entry of the uterus look convincing (e.g. Ahmad, , 1991. We can detect no pattern of association between the reported conformation of the terminal genitalia and other aspects of the morphology or host distribution, and can only suggest that the subject needs further active consideration. This distinction may be a basis for a future subdivision of the genus.

Host distribution
A critical component of the recognition of the validity of species of Opisthomonorcheides appears to lie in the nature of their host-specificity. In the host-parasite list ( The most interesting aspect of the host distribution of species of Opisthomonorcheides is the reported concentration of species in the black pomfret (Parastromateus niger), which has six recorded species and the silver pomfret (Pampus argenteus) with six species. In addition, the Chinese silver pomfret (P. chinensis) has two reported species. Despite the remarkable morphological similarity of Parastromateus and Pampus, these two genera are considered to belong to different families, the Carangidae and Stromateidae respectively; indeed, according to WoRMS (Bailly, 2008), the two families belong to separate suborders of the Perciformes. Two Opisthomonorcheides species (O. decapteri and O. pampi) are reported as being shared by these families. This distribution may be a striking case of morphological (and presumably dietary) convergence between the two groups of fishes, leading to the sharing of parasites. Alternatively, there may have been a series of misidentifications of some of the hosts. If the latter explanation is to hold, then we would predict that it is the stromateid hosts that have been misidentified, given that numerous additional carangids have been reported as hosts.
The credibility of a number of the remaining host records (and thus Opisthomonorcheides species) is O. nahhasi (Ahmad, 1985) 1 suspect. In our view, the evidence is clear that monorchiid species, in general, and species of Opisthomonorcheides, in particular, typically infect single or only closely related species, thus illustrating oioxenous or stenoxenous specificity. Accordingly, of all the species of Opisthomonorcheides, just two have been reported from multiple fish families. Opisthomonorcheides pampi has been reported from a stromateid, a carangid and a percichthyid. The sharing between a stromateid and a carangid is a special case, as discussed above. In our view, the record of O. pampi from a percichthyid is either not credible, accidental or not significant. We think that it is not plausible that a species should be shared by two such distantly related and ecologically different fishes but seemingly not by other, more closely related, sympatric hosts. The second species from different host families, O. decapteri, has been reported from multiple carangids, a stromateid, a macrourid and a lethrinid. In his discussion of the digenean parasites of macrourids, Bray (1995) cast doubt on the record from that family, pointing out that most other records are from pelagic carangids. Similarly, the record of O. decapteri from a lethrinid is based on a single highly macerated (as photographed) specimen with, apparently, two testes . We contend that the single specimen from a distantly related and ecologically different fish is not plausible.
Beyond the dominant records from carangids and stromateids, several species have been reported exclusively from other families: Apogonidae (O.  Despite the dramatic taxonomic differences between many of these families and the Carangidae and Stromateidae, many of the records and species appear valid given that they have been reported on the basis of multiple specimens. In this category are the reports of O. gibsoni, O. chaetodoni, O. upeneusi, O. madhavae, O. nahhasi, O. guptai and O. overstreeti (Ahmad, 1985) Madhavi, 2011. In contrast, O. engraulisi and O. simhai were described on the basis of just two specimens each from engraulids. The discussion of O. engraulisi does not mention O. simhai. The two species are strikingly similar and we consider the former a synonym of the latter. There is a precedent for such a pattern of host-specificity (rich in one family, scattered in others) among members of the Monorchiidae. Although species of Hurleytrematoides Yamaguti, 1954 occur overwhelmingly in chaetodontid fishes (22 species), a handful of species are reported from the Acanthuridae, Pomacanthidae, Siganidae and Tetraodontidae (see McNamara & Cribb, 2011).

Molecular data
It is our hope that the reporting of ITS2 rDNA sequences relating to O. pampi and O. ovacutus here O. yamagutii Gupta & Singh, 1990 will assist in the future recognition and delineation of species in this genus. The only other monorchiid genus for which there are sequence data available for multiple species is Hurleytrematoides. McNamara et al. (2014) successfully used ITS2 rDNA sequences (in parallel with mtCO1 sequences) to distinguish 16 species of Hurleytrematoides from chaetodontid and tetraodontid fishes in the Tropical Indo-west Pacific.
In that study, interspecific differences in the ITS2 region ranged from 8 to 84 bp. Analysis of the new data indicates that ITS2 sequences of sympatric O. pampi and O. ovacutus differed by 12 bp, a level which is consistent with the interspecific difference seen between species of Hurleytrematoides and in other trematode groups (Diaz et al., 2015;Nolan et al., 2016;Yong et al., 2016). We note that McNamara et al. (2014) reported differences up to 9 bp in the ITS2 region between samples of the morphologically distinctive species H. morandi McNamara & Cribb, 2011 from off the Great Barrier Reef and Palau. This was interpreted as geographical intraspecific variation; we thus caution that future comparisons of species of Opisthomonorcheides should expect, or at least allow for, the possibility of similar variation.