Introduction
The genus Clinostomoides was erected by Dollfus (Reference Dollfus1950) to accommodate a single adult collected from the esophagus of Ardea goliath sampled in the Belgian Congo (now Democratic Republic of the Congo – DRC). The type species, C. brieni Dollfus, Reference Dollfus1950, was distinguished from Clinostomum based on its large body size (30 mm), the genital complex in the posterior (rather than middle or across middle and posterior) third of body and the genital pore located ventral to the posterior testis (rather than lateral to the anterior testis). In the following years, metacercariae of C. brieni were described by Prudhoe (Reference Prudhoe1957) from Clarias lazera collected in Belgian Congo, by Manter and Pritchard (Reference Manter and Pritchard1969) from Clarias sp. from Rwanda, by Fischthal and Thomas (Reference Fischthal and Thomas1970) from C. senegalensis in Ghana, by Barson et al. (Reference Barson, Bray, Ollevier and Huyse2008) and by Jansen van Rensburg et al. (Reference Jansen van Rensburg, van As and King2013) from C. gariepinus sampled in Zimbabwe and Botswana, respectively. Outside the African continent, Mirzoeva (Reference Mirzoeva1981) described one adult of C. brieni collected from the esophagus of Ardea purpurea in Azerbaijan, and metacercariae of C. brieni were also reported in the Philippines (Arthur and Lumanlan-Mayo, Reference Arthur and Lumanlan-Mayo1997).
Additional species of Clinostomoides have been described in Central India, the first being C. dollfusi Agarwal, Reference Agarwal1958, followed by C. ophicephali (Tubangui and Masiluñgan, Reference Tubangui and Masiluñgan1935) Agarwal, Reference Agarwal1958 (transferred from genus Clinostomum), C. chauhani Pandey, Reference Pandey1971, C. rai Rai, Reference Rai1970, C. meerutensis Pandey and Tyagi, Reference Pandey and Tyagi1986, C. pandeyii Singh and Sharma, Reference Singh and Sharma1994 and C. baughi Pandey, Reference Pandey1998. However, all were erected based either on comparisons among these regional species or with the earliest species in the region, C. dollfusi. Most importantly, all, including C. dollfusi, were based on metacercariae. This is problematic because in erecting C. dollfusi, Agarwal (Reference Agarwal1958) compared metacercariae with the adult described by Dollfus (Reference Dollfus1950), not with metacercariae described by Prudhoe (Reference Prudhoe1957). Manter and Pritchard (Reference Manter and Pritchard1969) synonymized C. dollfusi with C. brieni, and their doubts about the morphological characters used to erect the junior species were confirmed by Fischthal and Thomas (Reference Fischthal and Thomas1970). However, subsequent work in India has not taken the latter studies into account and continues to treat as valid C. dollfusi and other species erected in comparison with it (e.g. Pandey and Agrawal, Reference Pandey and Agrawal2013). Finally, the species described by Dollfus has occasionally been reported as Clinostomum brieni, in lapsus calami. Prudhoe (Reference Prudhoe1957) and Douellou (Reference Douellou1992) used both ‘Clinostomum brieni’ and ‘Clinostomoides brieni’ as names for the same species, and Lio-Po et al. (Reference Lio-Po, Pascual and Santos1983) listed ‘Clinostomum brieni,’ but without taxonomic comment or support. Other than these isolated cases, the genus Clinostomoides and its type species C. brieni are widely considered valid (e.g. Kanev et al., Reference Kanev, Radev, Fried, Gibson, Jones and Bray2002).
A combination of molecular and morphological approaches is a useful way to resolve situations like this, as has already been shown in other clinostomids, i.e. Clinostomum Leidy, 1856 (Caffara et al., Reference Caffara, Locke, Gustinelli, Marcogliese and Fioravanti2011; Sereno-Uribe et al., Reference Sereno-Uribe, Pinacho-Pinacho, García-Varela and Pérez-Ponce de León2013), Euclinostomum Travassos, 1928 (Senapin et al., Reference Senapin, Phiwsaiya, Laosinchai, Kowasupat, Ruenwongsa and Panijpan2014; Caffara et al., Reference Caffara, Locke, Cristanini, Davidovich, Markovich and Fioravanti2016), Odhneriotrema Travassos, 1928 (Woodyard et al., Reference Woodyard, Rosser and Rush2017) and Ithyoclinostomum Witenberg, 1925 (Briosio-Aguilar et al., Reference Briosio-Aguilar, García-Varela, Hernández-Mena, Rubio-Godoy and Pérez-Ponce de León2018). However, few DNA sequences from Clinostomoides are currently available for comparison. Athokpam et al. (Reference Athokpam, Jyrwa and Tandon2014) provided rDNA sequences from C. brieni, but without supporting morphological information. Moreover, the identification of C. brieni by these authors was questioned by Briosio-Aguilar et al. (Reference Briosio-Aguilar, García-Varela, Hernández-Mena, Rubio-Godoy and Pérez-Ponce de León2018) because of the close relationship of its 28S sequence to data from Clinostomum. It is also relevant that the material sequenced by Athokpam et al. (Reference Athokpam, Jyrwa and Tandon2014) was from Heteropneustes in Northeastern India, rather than the region or hosts (central and southern Africa, Clarias, Ardea) where the genus originated and is better known.
The aim of this work was to provide a redescription of C. brieni metacercariae based on morphological and molecular analyses, following Matthews and Cribb (Reference Matthews and Cribb1998), and to provide an updated critical analysis of previously described species. To this end, we collected in localities and hosts in which we were likely to encounter the same species as Dollfus (Reference Dollfus1950) and Prudhoe (Reference Prudhoe1957), with the aim of characterizing the type species. Our results led us to transfer C. brieni to Clinostomum and to consider species of Clinostomoides from India as species inquirendae.
Materials and methods
Two metacercariae of Clinostomoides sp. were collected from Clarias gariepinus sampled at Phalaborwa barrage, Limpopo province (South Africa) and four from C. ngamensis sampled in the Democratic Republic of the Congo (one from Lake Tshangalele, Kapolowe Mission and three from Kiswishi River near Futuka Farm). All were recovered from the body cavity, cleaned in saline and preserved in 70% ethanol.
Total lengths of metacercariae were measured before cutting a small piece of the posterior end for molecular analyses. Morphometrics of hologenophores (sensu Pleijel et al., Reference Pleijel, Jondelius, Norlinder, Nygren, Oxelman, Schander, Sundberg and Thollesson2008) were taken after clarification with Amman's lactophenol and staining by Malzacher's method (Pritchard and Kruse, Reference Pritchard and Kruse1982). Line drawings were made with the aid of a drawing tube, and measurements are given in micrometres following Matthews and Cribb (Reference Matthews and Cribb1998). DNA was extracted from hologenophore subsamples using a PureLink Genomic DNA Kit (Invitrogen) following the manufacturer's protocol. Amplification of 18S and Internal transcribed Spacer 1 – 5.8S – Internal Transcribed Spacer 2 (ITS) rDNA employed protocols and primers of Gustinelli et al. (Reference Gustinelli, Caffara, Florio, Otachi, Wathuta and Fioravanti2010), cytochrome c oxidase I (CO1) mtDNA those of Moszczynska et al. (Reference Moszczynska, Locke, McLaughlin, Marcogliese and Crease2009).
Amplified products were resolved on a 1% agarose gel stained with SYBR Safe DNA Gel Stain in 0.5× TBE (Molecular Probes – Life Technologies). For sequencing of 18S, ITS and CO1, bands were excised and purified by NucleoSpin Gel and polymerase chain reaction Cleanup (Macherey-Nagel) and sequenced with an ABI 3730 DNA analyser at StarSEQ GmbH (Mainz, Germany). Contigs were assembled with Vector NTI Advance™ 11 software (Invitrogen) and sequences are published in GenBank under the following accession numbers: MH606186-90 (18S), MH238412-16 (ITS) and MH253044-48 (CO1).
Pairwise p-distances and models of nucleotide evolution (Bayesian Information Criterion) were calculated using MEGA 6.06 (Tamura et al., Reference Tamura, Stecher, Peterson, Filipski and Kumar2013). For trees constructed with Bayesian Inference (BI), in MrBayes 3.2.6 (Ronquist et al., Reference Ronquist, Teslenko, Van Der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012), nst = 2 + G was used for ITS and 18S, and GTR + G + I was used for CO1. The K2P + G-model was used for maximum likelihood (ML) analysis in MEGA of 18S and ITS rDNA while GTR + G + I was used for ML analysis of CO1 mtDNA.
The newly generated sequences of 18S, ITS and CO1 were aligned along with one or two representative sequences of Clinostomum species (C. complanatum, C. cutaneum, C. phalacrocoracis, C. tilapiae, C. philippinensis, C. marginatum, C. tataxumui, C. album, C. poteae, C. heluans, C. attenuatum, C. detruncatum, C. arquus, C. caffarae, C. cichlidorum) plus undescribed or unidentified species of Clinostomum (Locke et al., Reference Locke, Caffara, Marcogliese and Fioravanti2015; Caffara et al., Reference Caffara, Locke, Echi, Halajian, Benini, Luus-Powell, Tavakol and Fioravanti2017). Euclinostomum heterostomum (ITS: KP721422, CO1: KP721404), Odhneriotrema incommodum (ITS: MF766000, CO1: MF766003) and Ithyoclinostomum (ITS: MH159753, CO1: MH159752) were used as outgroup for the subfamily Clinostomatinae, while Tylodelphys immer (18S and ITS: MH521252; CO1: MH536513), Cyathocotyle prussica (18S and ITS: MH521249; CO1: MH536510), Schistosoma mansoni (18S: U65657; ITS: AY446082) as outgroup for Clinostomidae. All codon positions in the CO1 alignment were used in the analysis because of lack of evidence of nucleotide saturation (Iss = 0.237, Iss.c = 0.697, df = 472, P = 0; Xia et al., Reference Xia, Xie, Salemi, Chen and Wang2003; Xia and Lemey, Reference Xia, Lemey, Lemey, Salemi and Vandamme2009).
Results
Among five 18S rDNA sequences 1826–1877 bp in length obtained from African samples of C. brieni in the current study, there were four variable sites, all transitions, i.e. mean divergence 0.1%, range 0–0.2%. All variation was in two sequences from South Africa; three 18S sequences from Congo were identical. An 18S sequence (KF781300, 1907 bp) of Athokpam et al. (Reference Athokpam, Jyrwa and Tandon2014) from C. brieni from Heteropneustes fossilis in Manipur differed at 32 positions (1.7%) from another 18S sequence by the same authors (KF811009, 1859 bp) from the same host in Meghalaya (the latter sequence is not mentioned in the paper of Athokpam et al., Reference Athokpam, Jyrwa and Tandon2014). Variation in 18S between the five African C. brieni sequences and the two Indian isolates averaged 1.4% (range 0.5–2.6%). Phylogenetic analysis showed that 18S sequences from Indian and African C. brieni form a well-supported clade nested within Clinostomum species. The Clinostomum + C. brieni clade is also well supported, and comparatively deeply divergent from Euclinostomum. Variation among 18S sequences of Clinostomum spp. averaged 0.9% (range 0.2–1.5%) and in the Clinostomum + C. brieni clade, 18S variation averaged 1.1% (range 0–3.6%). All the highest divergence values (⩾1.8%) in the latter clade were associated with the unpublished C. brieni sequence KF811009. Variation between Euclinostomum and members of the Clinostomum + C. brieni clade averaged 2.8% (range 2.4–5.0%).
The five ITS rDNA sequences 1005–1028 bp in length from African C. brieni were identical to each other and to the 300-bp ITS2 sequence of C. brieni (KF781298) of Athokpam et al. (Reference Athokpam, Jyrwa and Tandon2014). The ITS of C. brieni varied by a mean of 5.7% (range 4.7–7.1%) from species of Clinostomum. Variation in ITS among species of Clinostomum s.s. was of similar magnitude: mean 4.9% (range 0.1–8.6%). In contrast, ITS variation among members of the genera Euclinostomum, Odhneriotrema and Clinostomum + Clinostomoides averaged 15.2% (range 13.9–16.3%).
The CO1 sequences of four specimens of C. brieni were identical but that of one specimen (MH253045, from C. gariepinus in South Africa) differed by 11% from the other four. This specimen did not differ morphologically from the other five C. brieni examined, and its 18S (MH606187) and ITS (MH238413) sequences were not similarly divergent (Figs 1 and 2). The CO1 of this specimen differed by 0.2% from Clinostomum morphotype 3 (KY865667, from the Amphilius uranoscopus in South Africa). The DNA from this specimen was amplified and sequenced an additional four times with the same results. The BI and ML trees were based on a 473 bp CO1 alignment and had little statistical support at deeper nodes, but both showed C. brieni within Clinostomum. The C. brieni specimen MH253045 grouped with Clinostomum sp. morphotype 3, while the other four C. brieni sequences form a monophyletic clade within the Old-World clade of Clinostomum species. The four monophyletic C. brieni sequences differ by mean 15.3% (range 13–19.5%) from other Clinostomum species. Interspecific CO1 variation in Clinostomum is similar, with mean 16.3% (range 3.5–22.1%). The mean intergeneric CO1 distances between members of Odhneriotrema, Clinostomoides + Clinostomum, Euclinostomum and Ithyoclinostomum is 19.8% (range 17.3–23.5%).
Evolutionary history inferred using BI (nst = 2 + G) from 18S rDNA of Clinostomum brieni n. comb. generated in this study (in bold) with data from other studies (22 nucleotide sequences, 1663 positions). Nodes are labelled with posterior probability in BI analysis and, after the slash, percent of bootstrap support in 1000 replicates in ML. SA, South Africa; DRC, Democratic Republic of the Congo.
Evolutionary history inferred using the BI analysis (nst = 2 + G) from ITS rDNA of C. brieni n. comb. generated in this study (in bold) with data from other studies (36 nucleotide sequences, 575 positions). Nodes are labelled posterior probability and, after the slash, with percent of bootstrap support in 1000 replicates in ML. SA, South Africa; DRC, Democratic Republic of the Congo.
Analyses of three molecular markers indicate Clinostomoides should be regarded as junior synonym of Clinostomum, as amended below. Tree topologies (Figs 1–3) show relatively deep divergence between Odhneriotrema, Euclinostomum and Clinostomum whereas Clinostomoides falls within a clade of Clinostomum species. Genetic distances between C. brieni and Clinostomum species are comparable with those within Clinostomum s.s. and inferior to distances among other clinostomid genera.
Evolutionary history inferred in BI analysis (GTR + G + I) from CO1 mtDNA of C. brieni n. comb. generated in this study (in bold) with data from other studies (37 nucleotide sequences, 473 positions). Nodes are labelled with posterior probability and, after the slash, percent of bootstrap support in 1000 replicates in separate ML analysis; an asterisk indicates a clade that was not recovered with ML. SA, South Africa; DRC, Democratic Republic of the Congo.
Clinostomum Leidy, 1856
(Synonym Clinostomoides Dollfus, Reference Dollfus1950)
Family Clinostomidae Lühe, 1901
Subfamily Clinostominae Lühe, 1901
Body medium to very large, linguiform, stout, convex dorsally and concave ventrally. Tegument smooth or with spines. Oral sucker may or may not be surrounded by collar-like fold when retracted. Ventral sucker muscular, well developed, always larger than oral sucker. Caeca long, simple, with more or less sinuous wall, particularly in anterior half of body, but lacking lateral branches or diverticula. Testes smooth or irregular in shape, in posterior half of body. Ovary intertesticular, to right of medial line. Vitelline follicles in lateral fields anteriorly, from the level of intestinal bifurcation or ventral sucker to posterior extremity, may remain lateral and extracaecal or become confluent posterior to genital complex. Uterus intercaecal, extending from Mehlis’ gland to fill part of total distance to ventral sucker. Genital pore anterior, lateral or posterior to testicular-ovary complex. Cosmopolitan.
Type species Clinostomum complanatum (Rudolphi, 1819).
Morphological description of Clinostomum brieni (Dollfus, Reference Dollfus1950) n. comb. (Fig. 4, Table 1) (based on five hologenophores and one paragenophore)
Line drawing of metacercaria of C. brieni n. comb. Scale bar = 1000 µm.
Morphological data of Clinostomum brieni [range (mean ± s.d.)]
(Synonym Clinostomoides brieni Dollfus, Reference Dollfus1950)
Body regularly elongated, narrow, tongue-shaped. Oral sucker small, with indistinct marginal limits. Pre-pharynx cup-shaped elongated, thick, muscular. Pharynx visible, muscular. Intestinal bifurcation anterior to ventral sucker, forming caecal shoulders before running laterally to ventral sucker to posterior end of body. Ventral sucker robust, larger than oral sucker, muscular, trilobed structure easily visible. Caeca provided with small lateral pockets becoming more digitated posteriorly to ventral sucker. Whole genital complex in posterior part of posterior third of body. Testes two, tandem, intercaecal, transversely elongated. Anterior testis bow-tie shaped. Posterior testis Y- to crescent shaped with anterior margin concave. Cirrus sac comma-shaped, thick walled, intertesticular dextral, from right posterior margin of anterior testis to anterior margin of posterior testis, genital pore opening in concave margin of posterior testis. Ovary small, intertesticular dextrally to cirrus sac, close to right margin of posterior testis.
Uteroduct emerging from ootype complex runs around left margin of anterior testis, ascending sinistrally with some undulation to slightly above metraterm before looping posteriorly on itself entering directly into the proximal part of uteroduct and opening into uterine sac. Uterine sac median elongate narrow, tip reaching the posterior part of middle third of body. Tegument armed with spines (8–11 µm) from posterior part of oral sucker to posterior end of body. Excretory bladder Y-shaped, postcaecal, arms extending anteriorly in extracaecal position. Excretory pore terminal.
Discussion
Our original aim was redescribe metacercariae of C. brieni and assess the relationship of the species with other clinostomids using DNA. Unexpectedly, the molecular data strongly indicate the species belongs within Clinostomum, which led us to amend the diagnosis of the genus to accommodate C. brieni n. comb. The genus Clinostomoides was limited to Afrotropic and Indo-Malayan regions, and most reports are from Clarias or Heteropneustes, which are closely related siluriform genera occurring in the same regions (Froese and Pauly, Reference Froese and Pauly2000; Hardman, Reference Hardman2005; Kushwaha et al., Reference Kushwaha, Kumar, Agarwal, Pandey, Nagpure, Singh, Srivastava, Joshi, Das, Sahoo and Jayasankar2015). The phylogenetic association of C. brieni with a clade of Old-World Clinostomum species (Figs 1–3) is consistent with a biogeographic pattern that continues to be observed as data accumulate from more species of Clinostomum (Locke et al., Reference Locke, Caffara, Marcogliese and Fioravanti2015; Pérez-Ponce de León et al., Reference Pérez-Ponce de León, García-Varela, Pinacho-Pinacho, Sereno-Uribe and Poulin2016; Rosser et al., Reference Rosser, Baumgartner, Alberson, Noto, Woodyard, King, Wise and Griffin2018), which adds further evidence that C. brieni belongs to Clinostomum.
One specimen we collected was morphologically indistinguishable and shared identical rDNA sequences with other C. brieni, but its CO1 (MH253045) was highly divergent, and nearly identical to Clinostomum morphotype 3, which Caffara et al. (Reference Caffara, Locke, Echi, Halajian, Benini, Luus-Powell, Tavakol and Fioravanti2017) obtained from mochokid and amphiliid catfishes in South Africa. We believe this can be most plausibly explained by hybridization. Both C. brieni and Clinostomum morphotype 3 infect siluriform second intermediate hosts in the same region, which are preyed upon by local ardeid definitive hosts, including the type host of C. brieni, A. goliath (Mock and Mock, Reference Mock and Mock1980). Other than this particular specimen, C. brieni and Clinostomum morphotype 3 appear to be distantly related (Figs 1 and 2). This argues against another possible explanation for mitochondrial haplotype sharing between C. brieni and Clinostomum morphotype 3, incomplete sorting among recently separated species. In any event, when viewed together with the highly distinctive morphology of C. brieni, both hybridization and incomplete lineage sorting suggest species belonging to a single genus, which supports our main taxonomic conclusion.
The most distinctive characters of Clinostomoides were the size of the adult (30 mm) described in A. goliath by Dollfus (Reference Dollfus1950), the extremely posterior position of the genital complex, and the position of the cirrus sac and genital pore within the genital complex, characters considered also in regional descriptions of metacercariae (Prudhoe, Reference Prudhoe1957; Manter and Pritchard, Reference Manter and Pritchard1969; Fischthal and Thomas, Reference Fischthal and Thomas1970; Jansen van Rensburg et al., Reference Jansen van Rensburg, van As and King2013). The most obvious distinction of Clinostomoides, its large size, is noteworthy in that the adult is known only from the largest of ardeids, A. goliath (Mock and Mock, Reference Mock and Mock1980). In light of the molecular evidence that Clinostomoides belongs within Clinostomum, the other characters may be considered as size-related allometric changes.
The clinostomid metacercariae we collected from Clarias species in South Africa and Democratic Republic of the Congo were morphologically consistent with previous descriptions of C. brieni in the same host and region (Prudhoe, Reference Prudhoe1957; Manter and Pritchard, Reference Manter and Pritchard1969; Fischthal and Thomas, Reference Fischthal and Thomas1970; Jansen van Rensburg et al., Reference Jansen van Rensburg, van As and King2013). The only inconsistency was the pharyngeal morphology. Fischthal and Thomas (Reference Fischthal and Thomas1970) reported a thick-walled, muscular pre-pharynx and very muscular pharynx (as observed in our specimens), while Prudhoe (Reference Prudhoe1957) reported the pharynx absent, Manter and Pritchard (Reference Manter and Pritchard1969) did not mention it, and Jansen van Rensburg et al. (Reference Jansen van Rensburg, van As and King2013) reported a short pre-pharynx and muscular pharynx. In our opinion, these structures are probably always present but not always visible.
As the genital complex provides reliable features for discriminating species of Clinostomum (i.e. Caffara et al., Reference Caffara, Locke, Echi, Halajian, Benini, Luus-Powell, Tavakol and Fioravanti2017; Sereno-Uribe et al., Reference Sereno-Uribe, García-Varela, Pinacho-Pinacho and Pérez-Ponce de León2018, see Table 2), it can also shed light on prior records of C. brieni. The posterior testis in most descriptions is crescent-shaped, and showed this form in two of six subjects we examined, but in four worms the posterior testis was Y-shaped, as also reported by Fischthal and Thomas (Reference Fischthal and Thomas1970). In metacercariae of Clinostomum, testes are more digitated than in pre-adults/adults (Ukoli, Reference Ukoli1966). The only description of the adult of C. brieni is that of Dollfus (Reference Dollfus1950), who reported testes similar to those later described in metacercariae, except for small marginal lobules in the adult organs. Thus, in this species of Clinostomum, developmental variation in the morphology of the testicular margin appears to be reversed (going from smooth to more digitated), although data are needed from additional adults to confirm this. The cirrus pouch (CP) in C. brieni is well developed and lies between the testes, at a variable distance from the posterior border of the anterior testis and touching the concave part of the posterior testis where the genital pore opens. This pattern has been observed in all previous descriptions of C. brieni except that of Fischthal and Thomas (Reference Fischthal and Thomas1970), in which the CP did not touch the posterior testis. Finally, in all metacercarial descriptions the uteroduct forms a similar loop devoid of eggs, which becomes filled with eggs in the adult (Dollfus, Reference Dollfus1950).
Morphological data and line drawings of the genital complex of C. brieni n. comb. and in species described from the genus Clinostomoides
BL, body length; BW, body; OSL, oral sucker length; OSW; oral sucker width; VSL, ventral sucker length; VSW, ventral sucker width; ATL, anterior testis length; ATW, anterior testis width; PTL, posterior testis length; PTW, posterior testis width; OL, ovary length; OW, ovary width; CPL, cirrus pouch length; CPW, cirrus pouch width; SA, South Africa, DRC, Democratic Republic of Congo; BC, Belgian Congo; (A), adult. The line drawings were based on figures in the original publications.
Molecular data indicate a single species (albeit with potential capacity to hybridize) of C. brieni among samples spanning approximately 1500 km of the known geographic range of this species, including the type region. Morphological differences among our specimens and those of prior regional accounts (Dollfus, Reference Dollfus1950; Prudhoe, Reference Prudhoe1957; Manter and Pritchard, Reference Manter and Pritchard1969; Jansen van Rensburg et al., Reference Jansen van Rensburg, van As and King2013) therefore likely represent intraspecific variation. This can provide a useful perspective for considering species of Clinostomoides described from the Indian subcontinent, the first of which was C. dollfusi, which Agarwal (Reference Agarwal1958) described from metacercariae from Clarias sp. and Heteropneustes (=Saccobranchus) sp. collected in Jabalpur. Agarwal (Reference Agarwal1958) emphasized body length (7.8–9.8 mm in the Indian metacercariae vs the 30 mm in adult holotype of C. brieni), the absence of lateral sacculations of the uterus, and distance between suckers. However, all these characters are in fact typical of metacercariae of C. brieni (Manter and Pritchard, Reference Manter and Pritchard1969; Fischthal and Thomas, Reference Fischthal and Thomas1970; Jansen van Rensburg et al., Reference Jansen van Rensburg, van As and King2013; present study), if not of the larger adult holotype. All other species of Clinostomoides in this region are from Heteropneustes. Clinostomoides chauhani Pandey, Reference Pandey1971, was described from the body cavity and viscera of H. fossilis collected in Lucknow, based on comparison with C. dollfusi and C. ophicephali, which were both synonymized with C. brieni by Manter and Pritchard (Reference Manter and Pritchard1969) and Fischthal and Thomas (Reference Fischthal and Thomas1970). The species C. chauhani is said to possess an aspinose body but spinose cuticle (Pandey and Agrawal, Reference Pandey and Agrawal2013). Clinostomoides rai was proposed by Rai (Reference Rai1970) for metacercariae from the intestine of Clarias batrachus collected in Mathura, on the basis of distance between suckers and limbs of uterus (Pandey and Agrawal, Reference Pandey and Agrawal2013). However, Pandey and Agrawal (Reference Pandey and Agrawal2013) appear to have mistranscribed morphological values (i.e. acetabulum 1.16 × 0.78 mm vs acetabulum at distance of 1.16 mm behind anterior end of body and 0.78 mm in diameter) and host tissues (muscle vs intestine) recorded by Rai (Reference Rai1970). Clinostomoides meerutensis Pandey and Tyagi, Reference Pandey and Tyagi1986 and C. pandeyii Singh and Sharma, Reference Singh and Sharma1994, were both created for metacercariae from the body surface of H. fossilis in Meerut based on a spinose tegument and ovary opposite to cirrus sac. Pandey and Kiran (Reference Pandey and Kiran2002) synonymized C. rai, C. meerutensis and C. pandeyii with C. dollfusi. Clinostomoides baughi Pandey, Reference Pandey1998 was described from metacercariae in skin near the operculum of H. fossilis in Lucknow, based only on comparisons with the species of Indian Clinostomoides mentioned above. In the descriptions of C. meerutensis, C. pandeyii and C. baughi, the ovary is described as opposite to the cirrus sac. However, to our knowledge the ovary in the genus Clinostomoides and/or Clinostomum is always on the same side as the CP. Line drawings in Pandey and Agrawal (Reference Pandey and Agrawal2013) appear to show specimens of the latter three species transposed, with the uteroduct in the right side of the body.
In our opinion, these species of Clinostomoides (C. dollfusi, C. rai, C. chauhani, C. meerutensis, C. pandeyii, C. baughi) described from India are species inquirendae because the morphological basis of each may be an artefact of development, beginning with the comparison of larval C. dollfusi with adult C. brieni and cascading through later descriptions. The characters considered in these studies vary with parasite development and may also be influenced by fixation and mounting (Manter and Pritchard, Reference Manter and Pritchard1969; Fischthal and Thomas, Reference Fischthal and Thomas1970). Until such species can be verified through morphological comparisons at equivalent stages of development, preferably with detailed accounts of the genital complex and with supporting molecular data, we consider only C. brieni to be valid. Essentially, this view follows Manter and Pritchard (Reference Manter and Pritchard1969) and Fischthal and Thomas (Reference Fischthal and Thomas1970). Further work may reveal additional species of Clinostomum with the Clinostomoides-morphotype on the Indian subcontinent or elsewhere, but currently this is supported with neither molecular (Figs 1 and 2) nor morphological data. Connectivity between Indian and African populations of C. brieni could be maintained by species of Ardea which occur in both regions (e.g. A. cinerea, A. purpurea, A. goliath, BirdLife International, 2018).
Author ORCIDs
Monica Caffara 0000-0003-3429-6121
Acknowledgements
The authors are grateful for the support of Professor A. Chocha Manda (Université de Lubumbashi, R.D. Congo) and for constructive suggestions made by two anonymous reviewers.
Financial support
Part of the research done in South Africa by AH was supported by the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation (NRF) of South Africa (Grant No. 101054). Any opinion, finding and conclusion or recommendation expressed in this material is that of the author(s) and the NRF does not accept any liability in this regard. SL was supported by the Puerto Rico Science Technology & Research Trust. MC, DB, TP and MLF received no specific grant from any funding agency, commercial or not-for-profit sectors.
Conflict of interest
None.
Ethical standards
Not applicable.
