Introduction
Species composition of Sphincteristomum Oshmarin, Mamaev & Parukhin, Reference Oshmarin, Mamaev and Parukhin1961 and Lobatotrema Manter, Reference Manter1963 within the Schistorchiinae Yamaguti, Reference Yamaguti1942 is controversial in regard to phylogenetics, systematic position, and differential morphological characteristics. Oshmarin et al. (Reference Oshmarin, Mamaev and Parukhin1961) erected Sphincteristomum for its type-species, Sphincteristomum acollum Oshmarin, Mamaev & Parukhin, Reference Oshmarin, Mamaev and Parukhin1961, collected from the intestine of the starry triggerfish, Abalistes stellatus (Anonymous) (Syn. Abalistes stellaris [Bloch & Schneider]) (Tetraodontiformes: Balistidae), from the Gulf of Tonkin off northern Vietnam. Manter (Reference Manter1963) established Lobatotrema for its type-species, Lobatotrema aniferum Manter, Reference Manter1963, collected from the intestine of an unidentified balistid off Fiji. Following a personal communication with Manter, Yamaguti (Reference Yamaguti1971) considered Lobatotrema as a synonym of Sphincteristomum based on a convergent morphological diagnosis between both genera, a proposal followed by Cribb and Bray (Reference Cribb and Bray1999), Cribb (Reference Cribb, Jones, Bray and Gibson2005), Abid-Kachour et al. (Reference Abid-Kachour, Mouffok and Boutiba2013), and Blend et al. (Reference Blend, Karar and Dronen2017). Additionally, L. aniferum was synonymized with S. acollum (see Yamaguti Reference Yamaguti1971), and while subsequent literature validated the synonymy, only Abid-Kachour et al. (Reference Abid-Kachour, Mouffok and Boutiba2013) considered L. aniferum to be a separate species within Sphincteristomum, designating it as Sphincteristomum aniferum (Manter, Reference Manter1963) Abid-Kachour, Mouffok & Boutiba, Reference Abid-Kachour, Mouffok and Boutiba2013. Molecular investigations of Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) on S. acollum and L. aniferum revealed them to be two species belonging to two genera, both possessing subtle morphological differences yet distinct host preferences. Accordingly, Lobatotrema was resurrected as a ‘cryptic genus’, phylogenetically distinguishable yet morphologically undifferentiated from Sphincteristomum. As a result, Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) revised and refined representative records to ensure the correct species. Accordingly, records of S. acollum have been restricted to A. stellatus, whereas those of L. aniferum have been restricted to triggerfishes within Balistoides Fraser-Brunner and Pseudobalistes Bleeker (Tetraodontiformes: Balistidae). By extension, Japanese records of S. acollum by Machida and Kuramochi (Reference Machida and Kuramochi1999) were defined as another record of L. aniferum (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023).
Presently, Lobatotrema is composed of only one representative species, L. aniferum. Records of this species sparked debate, particularly with the widening of its geographical range and comparative morphometrics (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023). Such parameters differentiated Japanese from Australian and New Caledonian records; however, Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) conserved the identity of these specimens within L. aniferum, awaiting further supportive evidence. Additionally, the molecular evidence of Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) differentiated two Australian populations of L. aniferum. While they interpreted molecular results as relating to geographical variation of a single species, supplementary species delimitation analyses – using the General Mixed Yule Coalescent (GMYC) and the Bayesian Poisson tree processes (bPTP) – by Mansour et al. (Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025) differentiated L. aniferum into two entities despite being morphologically undifferentiated and sympatric.
Besides S. acollum, representative species of Sphincteristomum include two poorly known species: Sphincteristomum nikolaevi Parukhin, Reference Parukhin1970 and Sphincteristomum mediterraneae Abid-Kachour, Mouffok & Boutiba, Reference Abid-Kachour, Mouffok and Boutiba2013. Parukhin (Reference Parukhin1970) presented a simplified description of S. nikolaevi for one specimen collected from the intestine of an unidentified triggerfish belonging to Rhinecanthus Swainson (Tetraodontiformes: Balistidae) from the Gulf of Aden and Red Sea. Abid-Kachour et al. (Reference Abid-Kachour, Mouffok and Boutiba2013) described S. mediterraneae for specimens from the intestine of the common pandora, Pagellus erythrinus (Linnaeus) (Spariformes: Sparidae), off Algeria in Oran Bay. The distinct host group and considerably distant/distinct locality of S. mediterraneae (a sparid fish in the East Atlantic Ocean [Mediterranean Sea]) are a quandary compared to records of Lobatotrema spp. and other Sphincteristomum spp. that are restricted to triggerfishes in the Indo-Pacific Ocean. On a broader level, this contradicts the paradigm of Indo-West Pacific distributions and representative hosts of the Schistorchiinae Yamaguti, Reference Yamaguti1942, which restricts records of schistorchiines to marine tetraodontiform and perciform fishes within the Indian and Pacific Oceans (Blend et al. Reference Blend, Karar and Dronen2017; Cribb Reference Cribb, Jones, Bray and Gibson2005; Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023; Pulis et al. Reference Pulis, Curran, Andres and Overstreet2014).
In light of the aforementioned controversial findings, our manuscript aims at addressing and revising Sphincteristomum (sensu lato) and the morphologically related Lobatotrema with their representative species, reassigning inconsistent species to their correct taxonomic positions, establishing criteria for defining Sphincteristomum (sensu stricto) and Lobatotrema, and examining each recognized species in regard to its geographic distribution, host-specificity, and characteristic features. Additionally, this manuscript represents an attempt to resolve ambiguities in records of L. aniferum (sensu lato) through thorough revision and detailed morphometrical comparisons coupled with available molecular evidence.
Materials and methods
Sample collection
Fishes were caught by small trawl off Safaga, Southern Hurghada, Egypt. Fish hosts were identified according to criteria established by Randall (Reference Randall1982) and Lieske et al. (Reference Lieske, Fiedler and Myers2004). Classification and authorities for fish follow FishBase (Froese and Pauly Reference Froese and Pauly2025). Fish were placed on ice in an adequate collection box and transported to the laboratory for examination. Dissections were conducted in accordance with the approved standard operating procedure (REC-FScSVU:03/12/23). For each fish, the alimentary canal from the oesophagus to the anus was removed as well as the surrounding peritoneal cavity and examined for endohelminths using an American Optical Forty Model 41–48 dissecting stereo microscope.
Parasite investigation
Digenea were relaxed in 1:3 unfiltered sea water to tap water as per Cribb and Bray (Reference Cribb and Bray2010), observed alive, flattened under a coverslip in 5% buffered formalin saline solution, preserved in 70% ethanol, stained with alum carmine, and mounted in a mixture of distyrene and a plasticizer dissolved in toluene-xylene (DPX). Schematic drawings were prepared using a Zeiss Universal compound microscope equipped with a camera lucida (Model PZO01852 10x) or a micro-projector. Measurements are in micrometres (μm) and expressed as ranges followed by mean ± standard deviation (SD). Comparative measurements were sourced from original species descriptions or re-descriptions, unless otherwise stated. Digenean identification was based on Cribb and Bray (Reference Cribb and Bray1999), Cribb (Reference Cribb, Jones, Bray and Gibson2005), Jones et al. (Reference Jones, Bray and Gibson2005), Blend et al. (Reference Blend, Karar and Dronen2017), and Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023). Ecological terms follow Bush et al. (Reference Bush, Lafferty, Lotz and Shostak1997).
Various museums provided photographs of the oral suckers/whole specimens of several species within the Schistorchiinae including the Harold W. Manter Laboratory (HWML), University of Nebraska, Lincoln, Nebraska, USA; the Meguro Parasitology Museum (MPM), Tokyo, Japan; the Natural History Museum (NHMUK), London, United Kingdom; the United States National Parasite Collection (USNM), Smithsonian National Museum of Natural History, Washington, DC, USA; and the Zoological Survey of India (ZSI), Kolkata, India. In addition, several specimens of deposited schistorchiines at the Parasitology Laboratory, Zoology Department, Faculty of Science, South Valley University (SVU) were examined. The oral suckers/whole worms from type and/or voucher specimens of the following species were examined: Paraschistorchis longivesiculurus (Hafeezullah, Reference Hafeezullah1981) Blend, Karar & Dronen, Reference Blend, Karar and Dronen2017 (ZSI W7282/1), Paraschistorchis seychellesiensis (Toman, Reference Toman1989) Blend, Karar & Dronen, Reference Blend, Karar and Dronen2017 (MPM 19144), Paraschistorchis stenosoma (Hanson, Reference Hanson1953) Blend, Karar & Dronen, Reference Blend, Karar and Dronen2017 (USNM 1338141; HWML 798, 1303, 1304, 1305), Paraschistorchis zancli (Hanson, Reference Hanson1953) Blend, Karar & Dronen, Reference Blend, Karar and Dronen2017 (USNM 1338142; HWML 1307, 1308), Plesioschistorchis callyodontis (Yamaguti, Reference Yamaguti1942) Blend, Karar & Dronen, Reference Blend, Karar and Dronen2017 (MPM23700; NHMUK 2017.9.28.10–11), Plesioschistorchis haridis (Nagaty, Reference Nagaty1957) Blend, Karar & Dronen, Reference Blend, Karar and Dronen2017 (USNM 1355361; HWML 1931, 2207, 2210; NHMUK 2017.9.28.1–9), Plesioschistorchis sigani (Yamaguti, Reference Yamaguti1942) Blend, Karar & Dronen, Reference Blend, Karar and Dronen2017 (MPM 23701), Schistorchis carneus Lühe, Reference Lühe1906 (ZSI W7286/1; NHMUK 2017.9.28.12–30; SVU 2017.11.Sc01–Sc07; SVU 2023.20.Sc01–Sc14) and Schistorchis skrjabini Parukhin, Reference Parukhin and Skjrabin1963 (ZSI W7284/1, W7285/1).
Molecular analysis
DNA extraction and amplification
Total genomic DNA was extracted from digenean trematodes using the Qiagen DNeasy® Blood and Tissue Kit (Cat. 69504). PCR amplifications were performed with a total volume of 50 μl consisting of 25 μl of Taq PCR Master Mix Kit (Qiagen, Cat. 201443), 0.5 μl of each primer, 1 μl of DNA template, and RNase-free water (Qiagen, Mat. 1012888) to achieve the final volume.
Two ribosomal genes were amplified; A partial fragment of the 28S rDNA (domains D1–D3; 1,200 bp) was amplified using the primers ZX-1 (forward: 5′-ACCCGCTGAATTTAAGCATAT-′3) (Bray et al. Reference Bray, Waeschenbach, Cribb, Weedall, Dyal and Littlewood2009) and 1500R (reverse: 5′-GCTATCCTGAGGGAAACTACT-′3) (Tkach et al. Reference Tkach, Littlewood, Olson, Kinsella and Swiderski2003) under thermocycling conditions specified by Anter et al. (Reference Anter, Georgieva, Gargouri and Kostadinova2015): 4 min denaturation at 94°C; 40 cycles at 95°C for 30 s, 54°C for 30 s, and 72°C for 2 min; with a final extension at 72°C for 7 min. The internal transcribed spacer 2 (ITS2 rDNA) was amplified using the primers GA1 (forward: 5’-AGAACATCGACATCTTGAAC-3’) (Anderson and Barker Reference Anderson and Barker1998) and ITS2.2 (reverse: 5’- CCTGGTTAGTTTCTTTTCCTCCGC-3’) (Cribb et al. Reference Cribb, Adlard and Bray1998) under the following thermocycling conditions: an initial cycle (95°C denaturation for 3 min, 45°C annealing for 2 min, 72°C extension for 90 s), followed by 4 cycles (95°C for 45 s, 50°C for 45 s, 72°C for 90 s), followed by 30 cycles (95°C for 20 s, 51°C for 20 s, 72°C for 90 s), then a final extension at 72°C for 5 min. The PCR amplicons were purified using the QIAquick® PCR Purification Kit (Qiagen, Cat. 28104) according to the manufacturer’s protocol and were sequenced directly using the ABI Prism Big Dye Terminator V.3.1 Cycle Sequencing Kit on an ABI 310 DNA stretch sequencer following the manufacturer’s recommendations using the same primers used for PCR amplification. Sequencing was carried out at Macrogen Inc., Seoul, South Korea. Both forward and reverse PCR reads were used for sequence assembly and editing as recommended by Blasco-Costa et al. (Reference Blasco-Costa, Cutmore, Miller and Nolan2016). GeneStudio ™ Professional Edition V. 2.2.0.0 was used to assemble, edit contiguous sequences, and generate consensus sequences.
Sequence alignments
Two new ribosomal sequences were generated for each of the partial sequences of the 28S rRNA and ITS2 rRNA. Each sequence was aligned with the available sequences of taxa within the Megaperidae Manter, Reference Manter1934, and selected outgroup taxa on GenBank (Table 1). Due to the limited reliability of the ITS2 marker for phylogenetic inference (Blasco-Costa et al. Reference Blasco-Costa, Cutmore, Miller and Nolan2016), an additional concatenated alignment consisting of sequences of partial 28S rDNA and complete ITS2 rDNA (Table 1) was constructed using Sequence Matrix v. 1.9 (Vaidya et al. Reference Vaidya, Lohman and Meier2011). A partition-homogeneity test with heuristic search was performed using PAUP v.4.0a169 (Swofford Reference Swofford2003) and a p-value > 0.05, indicating that the 28S rDNA and ITS2 rDNA sequences were congruent; thus, the two datasets could be analyzed together to get one phylogenetic tree. The three datasets were employed in an attempt to recognize more intra-schistorchiine phylogenetic relationships. The resultant alignments were refined manually using the MUSCLE algorithm as implemented in MEGA11 v.11.0.13 (Tamura et al. Reference Tamura, Stecher and Kumar2021). Regions with ambiguous alignment were removed, and the ends of each fragment were trimmed to a point where all sequences had started and finished. The numbers and percentage of variable sites for each dataset were estimated by computing pairwise distances using the ‘No. of differences’ model, with the inclusion of ‘Transitions + transversions’, substitutions, and ‘Pairwise deletions’ as the gaps/missing data treatment, within the MEGA11 v.11.0.13 software package (Tamura et al. Reference Tamura, Stecher and Kumar2021).
GenBank accession numbers for available sequence data of the Megaperidae Manter, Reference Manter1934, and outgroups used in the phylogenetic analyses
a Registered in GenBank as Sphincteristomum aniferum (Manter, Reference Manter1963) Abid-Kachour, Mouffok & Boutiba, Reference Abid-Kachour, Mouffok and Boutiba2013.
b The reference is an unpublished sequence from GenBank. More information is found in the references list of the text.
c Registered in GenBank as Atractotrema sigani Durio & Manter, Reference Durio and Manter1969.
Phylogenetic analysis
Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic analyses were applied to each of the three alignments: BI was performed with MrBayes v.3.2.6 (Ronquist et al. Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012), and ML analysis was executed with raxmlGUI v.2.0.10 (Edler et al. Reference Edler, Klein, Antonelli and Silvestro2021). The best nucleotide substitution model for BI settings was chosen using MrModeltest v.2.3 (Nylander Reference Nylander2004), and the Akaike Information Criterion (AIC) indicated that (SYM+G) was the best estimator for both 28S rRNA and ITS2 rRNA datasets. For ML settings, parameters of ModelTest-NG as an implemented algorithm in raxmlGUI v.2.0.10 (Edler et al. Reference Edler, Klein, Antonelli and Silvestro2021) selected (TVM-I+G4) and (GTR+I+G4) as the best substitution models according to the AIC for the 28S rRNA and ITS2 rRNA datasets, respectively. Nodal support for the ML analysis was calculated by performing a non-parametric bootstrap validation based on 1,000 pseudo-replicates. For BI analyses, four simultaneous Markov Chain Monte Carlo (MCMC) chains were sampled every 1,000 generations for a total of 1,000,000 generations; the first 3,000 samples were discarded as burn-in, and the average standard deviation of split frequencies was <0.01. Phylogenetic trees were visualized using FigTree v.1.4.4 (Rambaut Reference Rambaut2018). Posterior probability values [BI] <0.90 and bootstrap percentage values [ML] <70 were not reported.
Multivariate statistical analyses
For the analytical data interpretation, three statistical analyses were performed using IBM SPSS Statistics (V. 25) (IBM Corp. 2017): coefficient of variation (CV), correlation analysis (r), and cluster analysis.
The CV of each target measurement was calculated as the standard deviation (SD) divided by the mean value and presented as a percentage. Commonly used ranges for CV followed the classification of Pimentel-Gomes (Reference Pimentel-Gomes1985) as follows: CV<10% ‘low variability’; 10%≤CV≤20% ‘moderate variability’; CV>20% ‘high variability’. Metric features having a low coefficient of variation suggest less spread/dispersion, a high degree of consistency/uniformity, and relative stability in such measurements; hence, they are functional in differentiation and comparisons among related species.
Pearson correlation coefficient was calculated to determine the strength of relationships among the measurements (=variables) of target records and used to disclose the degree of dependency of one variable to the other. We investigated the correlation/relationship between body length and other measurements, given its importance as a growth indicator for the parasite. The strength of correlation was determined according to Lehman (Reference Lehman2005) as follows: r=zero ‘non-correlated’; r<0.20 ‘very weak’; 0.20≤r≤0.39 ‘weak’; 0.40≤r≤0.59 ‘moderate’; 0.60≤r≤0.79 ‘strong’; 0.80≤r<1 ‘very strong’; r=1 ‘perfect’. A two-tailed test of significance (p ‘probability value’) was estimated to determine the statistical significance of each measurement relative to others; p<0.05 is considered statistically significant. Measurements of high correlation with body length and probability <0.05 refer to a strong linear relationship, are considered statistically significant and unlikely to be a coincidence, and vice versa. Metric characters of strong correlation with body length though with a probability >0.05 indicate a relatively high chance that the observed correlation could have occurred by chance, hence making the character unreliable for comparisons; in addition, further measurements of such metric characters (i.e., more sampling) are suggested to determine the accurate range of their correlation with body length.
Cluster analysis (CA) was applied on morphologically closely related records of S. acollum, L. aniferum (sensu lato), and present specimens to identify groups of specimens with similar characteristics. Within CA, hierarchical agglomerative clustering (HAC) was employed to offer a flexible and insightful approach to exploring data, uncover hidden structures without requiring prior knowledge of the number of clusters, provide instinctive similarity relationships between any single sample and the entire dataset, and focus on merging clusters based on overall similarity rather than being heavily influenced by individual extreme values. Its typical illustration by a dendrogram offers valuable insights into how clusters are formed and how they relate to each other (see Ding and He Reference Ding and He2002; Jain Reference Jain2010; Rokach and Maimon Reference Rokach, Maimon, Rokach and Maimon2005). Inserted measurements in HAC were expressed as percentages relative to body length and/or ratios in all comparisons among target records (see Table S1). Data standardization was performed to prevent bias from feature scales, ensure that all features contribute equally to the distance calculations, and facilitate observing the relative importance of each feature in the clustering process, thus obtaining more reliable and meaningful clustering results (Xu and Wunsch Reference Xu, Wunsch, Xu and Wunsch2008). Ward’s method and squared Euclidean distance were applied to create compact, roughly equal-sized clusters, minimize within-cluster variance, and produce balanced cluster sizes. Furthermore, the two aforementioned parameters provide efficient computation, clear dendrogram interpretation, and determine statistically significant differences between the averages of study groups (see Everitt et al. Reference Everitt, Landau, Leese and Stahl2011; Murtagh and Legendre Reference Murtagh and Legendre2014).
Results
Sphincteristomum Oshmarin, Mamaev & Parukhin, Reference Oshmarin, Mamaev and Parukhin1961 (sensu stricto)
Taxonomic position
Superfamily Apocreadioidea Skrjabin, 1942
Family Megaperidae Manter, 1934
(Syns.: Apocreadiidae Skrjabin, 1942; Euryperidae Manter, 1933 nec Pascoe, 1870; Homalometridae Cable & Hunninen, 1942; Schistorchiidae Yamaguti, 1942; Sphincterostomatidae Yamaguti, 1937; Trematobrienidae Dollfus, 1950)
Subfamily Schistorchiinae Yamaguti, 1942
(Syn.: Sphincterostomatinae Yamaguti, 1958)
Genus Sphincteristomum Oshmarin, Mamaev & Parukhin, 1961 (sensu stricto)
Type-species:Sphincteristomum acollum Oshmarin, Mamaev & Parukhin, 1961
Other species: Sphincteristomum nikolaevi Parukhin, 1970
Diagnosis
(Modified after Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023). Body small, ovate to elliptical. Tegument spinous or unarmed. Eye-spot pigment dispersed in forebody. Forebody short, about a quarter of body length. Pre-oral lobe present. Oral sucker large, evenly round in outline to subglobular, occasionally short and pyriform, subterminal, glandular/cellular without circumoral lobes; partial U-shaped sphincter half encircles oral aperture postero-ventrally. Ventral sucker pre-equatorial, sessile, muscular, globular, half size of oral sucker. Pre-pharynx absent. Pharynx nearly globular to broadly oval, non-papillate, weakly developed, as large as ventral sucker or slightly larger. Oesophagus not evident. Intestinal bifurcation posterior to mid-forebody level, immediately anterior to ventral sucker or separated by short distance. Caeca two, long, open via separate ani at posterior end of body. Testes two, intercaecal, median, smooth to slightly indented or distinctly lobed, contiguous, elliptical to irregular, tandem to opposite or symmetrical, post-ovarian, in anterior hindbody. Post-testicular region large, more than two-fifths of body length. Seminal vesicle saccate, unipartite, dextral and proximate to ventral sucker and right cecum. Pars prostatica tubular, short, forms short hermaphroditic duct which itself extends antero-dorsal to ventral sucker. Prostatic cells few, free in parenchyma. Genital atrium indistinct. Genital pore ventro-submedian (sightly sinistral) to median, immediately anterior to or just overlapped by anterior margin of ventral sucker. Ovary subglobular to transversally oval, smooth, in anterior hindbody, median, pre-equatorial, pre-testicular, contiguous with anterior testis. Vitellarium follicular; distributed from near posterior extremity along lateral margins to posterior margin of oral sucker, confluent in post-testicular region. Follicles profuse, dorsal and lateral to caeca. Uterus short, pre-ovarian, intercaecal, extends between level of ovary and genital pore. Metraterm indistinct. Eggs few, operculate, oval, thin. Excretory vesicle I-shaped, moderately wide, median, extends anteriorly to posterior testis; excretory pore terminal to ventrally subterminal. In intestine of balistid fishes (Abalistes Jordan & Seale; Sufflamen Jordan; Rhinecanthus) in Indo-Pacific Ocean.
Remarks
Distinct environmental conditions distinguish the Indo-Pacific from the Mediterranean Sea, including warmer waters, lower salinity, an open basin more influenced by oceanic currents and atmospheric patterns, lower evaporation rates, and more complex circulation patterns affected by monsoons and equatorial currents. The Indo-Pacific Ocean hosts a wider array of marine species (Castro and Huber Reference Castro and Huber2010; Stewart Reference Stewart2008). The Mediterranean Sea is more enclosed suggesting a more unique assemblage of marine species and related Digenea. Investigations by Blend et al. (Reference Blend, Karar and Dronen2017), Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023), Mansour et al. (Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025), and the present phylogenetic analyses (see below) of the Schistorchiinae highlight the significant role of host specificity in species/genera recognition and clarify that each genus of schistorchiine, at the very least, is restricted to a limited range of environmentally/taxonomically associated hosts and closely related/convergent geographical distributions. Hence, reporting a taxon of Sphincteristomum from sparid fishes in the Mediterranean Sea (see Abid-Kachour et al. Reference Abid-Kachour, Mouffok and Boutiba2013) could represent a possible misidentification compared with several records of Sphincteristomum spp. from balistid fishes in the Indo-Pacific Ocean (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023; Oshmarin et al. Reference Oshmarin, Mamaev and Parukhin1961; Parukhin and Chikunova Reference Parukhin and Chikunova1964; Parukhin Reference Parukhin1970, Reference Parukhin1989; Present study), the latter of which are further investigated herein to determine their taxonomic status. Accordingly, we have restricted the concept of Sphincteristomum to species collected from balistid fishes in the Indo-Pacific Ocean only: S. acollum (off Vietnam) and S. nikolaevi (Gulf of Aden and the Red Sea).
Although Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) described the oral sucker of S. acollum as roughly pyriform, their description was based on a single incomplete specimen (i.e., missing part of its posterior end), making observation of intra-specific variation in oral sucker shape impossible. Comparatively, the original records of Oshmarin et al. (Reference Oshmarin, Mamaev and Parukhin1961) and S. nikolaevi (Parukhin Reference Parukhin1970; Present study) confirm that the oral sucker has a distinctive shape: evenly round in outline, neither constricted anteriorly nor pyriform. Hence, we believe that the incidental presence of a short, pyriform shaped oral sucker in a partially damaged sample represents the exception rather than the rule. Further, Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) diagnosed the ovary position in Sphincteristomum spp. as dextral or almost median in the anterior hindbody. With the exclusion of the Mediterranean taxon of Sphincteristomum (see Abid-Kachour et al. Reference Abid-Kachour, Mouffok and Boutiba2013), we find that the ovary in records of S. acollum (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023; Oshmarin et al. Reference Oshmarin, Mamaev and Parukhin1961) and S. nikolaevi (see Parukhin Reference Parukhin1970; Present study) has a pre-equatorial median position in the anterior hindbody. Accordingly, we have restricted the diagnosis of Sphincteristomum to species possessing an oral sucker of an evenly round to occasionally short pyriform shape as well as a pre-equatorial, median ovary located within the anterior hindbody.
Species recognized within Sphincteristomum (sensu stricto)
Sphincteristomum acollum Oshmarin, Mamaev & Parukhin, Reference Oshmarin, Mamaev and Parukhin1961
Illustrations of recognized taxa within Sphincteristomum Oshmarin, Mamaev & Parukhin, Reference Oshmarin, Mamaev and Parukhin1961 (sensu stricto). (a) Sphincteristomum acollum Oshmarin, Mamaev & Parukhin, Reference Oshmarin, Mamaev and Parukhin1961 modified after Oshmarin et al. (Reference Oshmarin, Mamaev and Parukhin1961, Figure 3), ventral view. (b) Sphincteristomum nikolaevi Parukhin, Reference Parukhin1970 modified after Parukhin (Reference Parukhin1970, Figure 1), ventral view. Scale bars: (a, b) 500μm.
Records
1. Oshmarin et al. (Reference Oshmarin, Mamaev and Parukhin1961); 2. Parukhin and Chikunova (Reference Parukhin and Chikunova1964); 3. Parukhin (Reference Parukhin1989); 4. Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023).
Remarks
Reviews of Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) clarified the oioxenous nature of S. acollum as it was restricted only to a single host species, A. stellatus. This is consistent with our host-parasite data investigations and species delimitation analyses (see below). The most notable characteristic of S. acollum within the restricted concept of Sphincteristomum is the asymmetrical, deeply lobed, tandem testes and its oioxenous nature.
Sphincteristomum nikolaevi Parukhin, Reference Parukhin1970
Illustrations of Sphincteristomum nikolaevi Parukhin, Reference Parukhin1970 from the bluethroat triggerfish, Sufflamen albicaudatum (Rüppell). (a) Adult whole mount, voucher specimen, ventral view. (b) Terminal genitalia, dorsal view. (c) Proximal female genital system, dorsal view. (d) Elliptical eggs. Scale bars: (a) 500μm; (b, c) 200μm; (d) 100μm.
Taxonomic summary
Hosts: Bluethroat triggerfish, Sufflamen albicaudatum (Rüppell) (Balistidae); Picasso triggerfish, Rhinecanthus assasi (Forsskål) (Balistidae).
Locality: Northern Red Sea, off Safaga, Southern Hurghada, Egypt.
Collection dates: S. albicaudatum (12/Jun/2022 and 7, 23/Aug/2022); R. assasi (25/May/2022 and 23/Aug/2022).
Site of infection: Intestine.
Prevalence: 4 out of 9 S. albicaudatum (44.4% infected); 2 out of 7 R. assasi (28.6% infected).
Intensity: 1–2 worms in S. albicaudatum; 1–2 worms in R. assasi.
Mean intensity: 1.5 (6/4) worms in S. albicaudatum; 1.5 (3/2) worms in R. assasi.
Relative density/abundance: 0.67 (6/9) worms in S. albicaudatum; 0.43 (3/7) worms in R. assasi.
Deposited specimens: Parasitology Laboratory, Zoology Department, Faculty of Science, South Valley University; Collector YFMK and AM, Vouchers (n = 7) (SVU) 2025.22.Sn01–Sn07.
Records: 1. Parukhin (Reference Parukhin1970); 2. Present study.
Supplemental description
[Based on 6 mature specimens]. Worms white in life. Body delicate, ovate to sub-elliptical with almost parallel margins; maximum width near equatorial region. Tegument thin, aspinose, smooth. Anterior extremity broadly rounded; posterior extremity rounded or attenuated. Forebody shorter than hindbody, slightly narrows near anterior end; hindbody larger, gradually widens near midbody, then gradually narrows towards posterior extremity. Pre-oral lobe short. Oral sucker spherical, glandular, ventro-subterminal, of ‘complex’ type, without circumoral lobes. Mouth conspicuous, rounded, directed antero-ventrally; surrounded by strong semi-circular muscle fibres that sometimes cover mouth aperture; partial U-shaped muscular sphincter half-encircles oral aperture postero-ventrally. Ventral sucker pre-equatorial, spherical, sessile, unspecialized, about one-third size of oral sucker, at junction of first and second quarters of body. Suckers separated by a short distance to almost contiguous. Pre-pharynx absent. Pharynx transversally elliptical or compressed, weakly developed, not papillate, equal or slightly smaller than ventral sucker, overlapped by oral and occasionally by ventral sucker. Oesophagus not evident. Intestinal bifurcation at end level of forebody, midway between suckers, contiguous to ventral sucker or slightly separated by short distance. Caeca two, simple, wider anteriorly, slightly arcuate posteriorly, subequal in length, extend laterally to lateral margins, terminate at posterior extremity in small, separate ani; anus lateral, slit-like, narrow, subterminal.
Testes two, smooth, sub-elliptical to sub-oval, subequal in size, symmetrical, contiguous laterally, occupy second fifth of body. Post-testicular space occupies approximately two-thirds of body. Seminal vesicle saccate, large, transversally sub-elliptical, unipartite, majority dextral to midline, distal portion narrower, confined to area between ovary, ventral sucker, right testis, and right cecum. Pars prostatica tubular, short, thin-walled, passes parallel to anterior margin of ventral sucker to terminate in hermaphroditic duct. Prostatic cells few, free in parenchyma, surround pars prostatica and hermaphroditic duct. Genital pore median, opens immediately anterior to ventral sucker.
Ovary oval, smooth, pre-equatorial, in posterior portion of anterior third of body, median, overlaps ventral sucker dorsally, either entirely pre-testicular or sometimes overlaps anterior margins of testes ventrally. Seminal receptacle and oötype complex not observed. Vitellarium follicular, extends along lateral margins from as far anterior as level of pharynx to near posterior extremity; overlaps lateral and posterior margins of testes, confluent in post-testicular region. Vitelline follicles numerous, small, sub-spherical to oval; lateral and ventral to caeca. Right and left transverse vitelline ducts unite immediately posterior to ventral sucker; small median vitelline reservoir opens into oötype. Uterus pre-ovarian, short, saccular, intercaecal, in posterior forebody; restricted to area between ovary, seminal vesicle, left testis, left caecum, and genital pore; dorsally overlaps left edge of ventral sucker and extends antero-sinistrally around left margin of ventral sucker. Post-uterine distance very long, about last two-thirds of body. Metraterm tubular, short, wide, runs a short distance parallel to left and anterior margin of ventral sucker, then unites with distal portion of pars prostatica to form hermaphroditic duct at mid-anterior margin of ventral sucker; duct itself cylindrical, short, extends antero-dorsally to ventral sucker. Eggs few, operculated, oval, thin-shelled, non-filamented.
Excretory vesicle I-shaped, median, wider posteriorly, extends to near anterior end of second third of body, close behind testes. Excretory pore terminal.
Variability in adult specimens
Applying the coefficient of variation (CV) on morphometric features of S. nikolaevi reveals a substantial dispersion in variability, from less variable, moderate, to more variable as shown in Table 2. Body width and ovary width as measurements and percentages of body length show a low CV of a strong, significant correlation with body length (r≥0.98, p<0.05). Other metric characters exhibit low CV with non-significant correlation to the body length (r≤0.41, p>0.05), incorporating measurements of forebody length, oral sucker size, right testis width, pre-bifurcal distance, pre-genital pore distance, pre-ovarian distance, and egg width in addition to ovary length as a percentage of body length. Some metric characters with low CV show a strong correlation with body length (r≥0.59), however, insignificant (p>0.05). These include measurements of ventral sucker length, left testis width, pre-vitelline distance, pre-testicular distance, and egg length as well as pre-vitelline distance, pre- and post-testicular distance, and post-uterine distance as percentages of body length, and sucker length ratio. Out of 13 morphometric characters with moderate CV, the ratio of pre-genital pore distance to body length only shows a significant correlation with body length (r=-0.88, p<0.05). Concerning morphometric characters with high CV, measurements and ratios of distance from the intestinal bifurcation to the ventral sucker is notably the largest (CV=200). Furthermore, measurement of distance from ventral sucker to anterior testis, post-testicular distance, and post-uterine distance in addition to percentages of ventral sucker length relative to body length exhibit a significant correlation with body length (r≥0.95, p≤0.05).
Measurements, morphometric percentages, and morphometric ratios of taxa within Sphincteristomum Oshmarin, Mamaev & Parukhin, Reference Oshmarin, Mamaev and Parukhin1961 (sensu lato)
--- = Neither given in original description nor available from published illustration(s).
[ ] Values outside the original description as a result of either the potential inaccuracy of original calculations &/or values of scale bars.
() Mean values of the target measurement followed by the standard deviation.
a Starry triggerfish, Abalistes stellatus (Anonymous) (Syn. Abalistes stellaris [Bloch & Schneider]).
b Undefined triggerfish belonging to Rhinecanthus Swainson.
c Picasso triggerfish, Rhinecanthus assasi (Forsskål).
d Bluethroat triggerfish, Sufflamen albicaudatum (Rüppell).
e Common pandora, Pagellus erythrinus (Linnaeus).
f Proportion of body length; range values are given first followed by the mean values.
g Calculated from measurements given in the original description.
h Calculated from the illustrations of the original description (Oshmarin et al. Reference Oshmarin, Mamaev and Parukhin1961, Figure 3; Parukhin, Reference Parukhin1970, Figure 1; Abid-Kachour et al. Reference Abid-Kachour, Mouffok and Boutiba2013, Figures 2b, 3a, b, 4a–c).
i Measurements of the type-specimen of S. acollum as given in the original description.
j CV, coefficient of variation; r, Pearson correlation coefficient; p, Probability value.
k We consider this species a species inquirendum.
Remarks
Based on parasitizing the alimentary canal of marine fishes, possessing a mouth at the anterior extremity, the presence of both suckers and a pharynx, a hermaphroditic duct, the absence of a pre-pharynx and hermaphroditic sac, unarmed tegument, an I-shaped excretory vesicle and testes in the hindbody, our specimens are identified within the Megaperidae and the Apocreadioidea (Blend et al. Reference Blend, Karar and Dronen2017, Reference Blend, Karar and Dronen2024; Bray Reference Bray, Jones, Bray and Gibson2005a, Reference Bray, Jones, Bray and Gibson2005b, Reference Bray, Bray, Gibson and Jones2008; Cribb Reference Cribb, Jones, Bray and Gibson2005; Cribb and Bray Reference Cribb and Bray1999; Jones Reference Jones, Jones, Bray and Gibson2005). Having a genital pore anterior to the ventral sucker and an oral sucker with a partial U-shaped sphincter half encircling the oral aperture postero-ventrally, our specimens are consistent with the diagnosis of the Schistorchiinae (Blend et al. Reference Blend, Karar and Dronen2017; Cribb Reference Cribb, Jones, Bray and Gibson2005, Cribb and Bray Reference Cribb and Bray1999). Our material keyed out to Sphincteristomum on the basis of having the following combination of morphological features: two testes, caeca open independently via separate ani, a highly glandular oral sucker, and a balistid host (Blend et al. Reference Blend, Karar and Dronen2017; Cribb Reference Cribb, Jones, Bray and Gibson2005; Cribb and Bray Reference Cribb and Bray1999; Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023). With identical morphological features to those of Parukhin (Reference Parukhin1970) and a similar geographic locality (Red Sea) as well as possessing similar measurements, morphometric percentages and ratios for observed features (Table 2), we identify our material as S. nikolaevi.
A review of the previous record of Sp. nikolaevi and comparison with our specimens reveal a consistency in morphological features without considerable intra-specific variation. Likewise, morphometric percentages and ratios are similar except that our specimens exhibit higher values in length of both suckers and pre-bifurcal and pre-vitelline length as percentages of body length, as well as an increase in ratios of pharynx/oral sucker length, pharynx/ventral sucker length, and mean testes length/ovary length. In contrast, ratios of distance between suckers and distance from intestinal bifurcation to ventral sucker relative to body length as well as sucker ratio are smaller than those of Parukhin (Reference Parukhin1970). All aforementioned variations in morphometric percentages and ratios exhibit a moderate to high coefficient of variation, which weakens reliance upon such features as important differential characters—hence, insufficient to demonstrate non-conspecifity with Sp. nikolaevi. These slight variations might be due to host-induced variability (unidentified triggerfish, Rhinecanthus sp. vs Su. albicaudatum and R. assasi) and the different treatments used in parasite preparation.
In comparison with S. acollum, S. nikolaevi is characterized by symmetrical, smooth, opposite testes, a shorter testicular zone, and a longer post-testicular distance exceeding half the body length, and it represents a stenoxenous species as it was collected from phylogenetically related hosts (see Parukhin Reference Parukhin1970; Present study; Table 2). In contrast, S. acollum possesses asymmetrical, deeply lobed, tandem testes, a longer testicular zone (about twice that of S. nikolaevi), and a shorter post-testicular distance (less than half the body length), and it is an oioxenous species (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023; Table 2; Oshmarin et al. Reference Oshmarin, Mamaev and Parukhin1961).
To our knowledge, R. assasi is known to harbor three digenean parasites from the northern Red Sea: an acanthocolpid, Pseudolepidapedon balistis Manter, Reference Manter1940 (Khalifa et al. Reference Khalifa, Hassan, Mohamadain and Karar2015); a gyliauchenid, Flagellotrema convolutum Ozaki, Reference Ozaki1936 (Karar et al. Reference Karar, Blend, Khalifa, Hassan, Mohamadain and Dronen2019); and an opecoelid, Gaevskajatrema balistes Karar, Blend, Mohamadain, Hassan, Khalifa & Dronen, 2020 (Karar et al. Reference Karar, Blend, Mohamadain, Khalifa and Dronen2020). We found no megaperid/schistorchiine record from R. assasi, so the finding of Sp. nikolaevi herein represents a new host record for megaperids/schistorchiines, and Sphincteristomum. Concerning Su. albicaudatum, no helminth infection has been reported; accordingly, the finding of Sp. nikolaevi herein represents the first record of a helminth infecting Su. albicaudatum and widens the range of host variation of Sp. nikolaevi.
Lobatotrema Manter, Reference Manter 1963
Taxonomic position
Superfamily Apocreadioidea Skrjabin, 1942
Family Megaperidae Manter, 1934
(Syns.: Apocreadiidae Skrjabin, 1942; Euryperidae Manter, 1933 nec Pascoe, 1870; Homalometridae Cable & Hunninen, 1942; Schistorchiidae Yamaguti, 1942; Sphincterostomatidae Yamaguti, 1937; Trematobrienidae Dollfus, 1950)
Subfamily Schistorchiinae Yamaguti, 1942
(Syn.: Sphincterostomatinae Yamaguti, 1958)
Genus Lobatotrema Manter, 1963
Type-species: Lobatotrema aniferum Manter, 1963
Other species: Lobatotrema droneni n. sp.
Diagnosis
(Modified after Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023). Body small, elongate. Tegument spinous anteriorly or aspinous. Eye-spot pigment dispersed in forebody. Forebody short, about a one-quarter of body length. Pre-oral lobe present. Oral sucker quite large, elongate ovoid to pyriform, subterminal, highly glandular/cellular without circumoral lobes; partial V-shaped sphincter half encircles oral aperture postero-ventrally. Ventral sucker pre-equatorial, sessile, muscular, globular, less than one-half size of oral sucker. Pre-pharynx absent or very short. Pharynx subquadrate to broadly oval, non-papillate, weakly developed, slightly smaller than ventral sucker. Oesophagus absent. Intestinal bifurcation immediately posterior to pharynx, anterior to ventral sucker. Caeca two, long, open via separate ani at posterior end of body. Testes two, intercaecal, median, smooth to indented, contiguous, elliptical to irregular, tandem, in anterior hindbody. Post-testicular region large, exceeds one-third of body length. Seminal vesicle saccate, unipartite, overlapped by or dextral to ventral sucker. Pars prostatica tubular, combined with distal portion of metraterm to form short hermaphroditic duct which itself extends antero-dorsal to ventral sucker. Prostatic cells few, free in parenchyma. Genital atrium indistinct. Genital pore ventro-submedian (sightly sinistral), post-bifurcal, immediately anterior to ventral sucker. Ovary subglobular to transversally oval, smooth, in anterior hindbody, dextro-submedian, pre-testicular, contiguous with anterior testis. Vitellarium follicular; distributed from near posterior extremity along lateral margins to posterior forebody, confluent in post-testicular region; follicles profuse, small, dorsal and lateral to caeca. Uterus short, pre-/para-ovarian, intercaecal. Metraterm indistinct. Eggs few, operculate, oval, thin. Excretory vesicle I-shaped, moderately wide, median, extends anteriorly to beginning of post-testicular level; excretory pore terminal to ventrally subterminal. In intestine of balistid fishes (Balistoides, Pseudobalistes) in Indo-Pacific Ocean.
Remarks
Recent investigations of Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) resurrected Lobatotrema independent from Sphincteristomum on the basis of obvious phylogenetic divergence coupled with morphology/host group-based limited distinction. Phylogenetically, taxa of Lobatotrema showed differences in genetic distance in cox1 mtDNA, ITS2 rDNA, and 28S rDNA sequence data compared to that of Sphincteristomum, resulting in a phylogenetic separation of both genera into separate clades (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023; Mansour et al. Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025; Results below). Morphology/host group-based limited distinction included a V-shaped oral sphincter, smooth to slightly indented testes, a relatively thinner body, a slightly higher value in ventral sucker/oral sucker width ratio, and restriction to balistid fishes of Balistoides and Pseudobalistes. However, morphometric convergences alongside the belief in the absence of reliable genus-level morphological distinctions have caused confusion in the generic diagnosis of Lobatotrema. Accordingly, Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) considered Lobatotrema a ‘cryptic genus’, phylogenetically distinguished but morphologically undifferentiated from the concept of Sphincteristomum.
The present study is consistent with results of Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) except for lack of fundamental morphological distinction between Lobatotrema and Sphincteristomum. Our phylogenetic results exhibit a distinct difference between Lobatotrema and Sphincteristomum – the shape of the oral sphincter (Lobatotrema spp. possess a V-shaped oral sphincter vs a U-shaped oral sphincter in Sphincteristomum spp.) – which in turn, can be used for differentiating between schistorchiine genera. Additionally, Lobatotrema spp. possess a dextral ovary which differs from a median ovary in Sphincteristomum spp.
Species recognized within Lobatotrema Manter, 1963
Lobatotrema aniferum Manter, Reference Manter1963 (sensu lato)
Illustrations of recognized taxa within Lobatotrema Manter, Reference Manter1963. (a) Lobatotrema aniferum Manter, Reference Manter1963 (sensu lato) modified after Manter (Reference Manter1963, Figure 8), ventral view. (b) Holotype of Lobatotrema droneni n. sp. from the yellowmargin triggerfish, Pseudobalistes flavimarginatus (Rüppell), ventral view. Scale bars: (a) 500μm; (b) 300μm.
(Syns. Sphincteristomum aniferum [Manter, 1963] Abid-Kachour, Mouffok & Boutiba, Reference Abid-Kachour, Mouffok and Boutiba2013; Sphincteristomum acollum sensu Machida & Kuramochi [1999] nec Oshmarin, Mamaev & Parukhin, Reference Oshmarin, Mamaev and Parukhin1961)
Records
1. Manter (Reference Manter1963); 2. Machida and Kuramochi (Reference Machida and Kuramochi1999); 3. Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023).
Remarks
Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) redescribed L. aniferum from the intestine of three balistids from three close localities: the titan triggerfish, Balistoides viridescens (Bloch & Schneider), and the yellowmargin triggerfish, Pseudobalistes flavimarginatus (Rüppell) (Tetraodontiformes: Balistidae), both off Lizard Island, northern Great Barrier Reef (GBR); and the yellow-spotted triggerfish, Pseudobalistes fuscus (Bloch & Schneider) (Tetraodontiformes: Balistidae), off Îlot Goéland, New Caledonia and Ningaloo Reef, Western Australia. Collective morphological, molecular, and host distribution results strongly clarified that described specimens of L. aniferum by Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) align with the original characterization by Manter (Reference Manter1963) and are distinctly separate from S. acollum. These results demonstrated the following variations: (i) the form of oral sphincter from U-shaped for S. acollum vs V-shaped for L. aniferum, (ii) the degree of testis lobation from distinctly lobed for S. acollum vs smooth to slightly indented for L. aniferum, (iii) body width from broader for S. acollum vs thinner for L. aniferum, (iv) distinctive genetic distance between the two species in cox1 mtDNA, ITS2 rDNA, and 28S rDNA sequences, (v) a phylogenetic divergence of the two species within non-sister clades, and (vi) oioxenicity of S. acollum vs stenoxenicity of L. aniferum within balistid fishes of Balistoides and Pseudobalistes spp. (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023). However, convergence in several morphometric measurements overlap in morphological descriptions of some internal features between the two species, and unsubstantial disparities among examined specimens caused absences of reliable genus-level morphological distinctions, compared to the unambiguous molecular distinction. Hence, Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) concluded a limited morphological and ecological differentiation between L. aniferum and S. acollum, but substantial molecular discrimination at the genus level.
Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) reassigned Japanese records of S. acollum as L. aniferum based on matching morphology and shared host-parasite data (see Machida and Kuramochi Reference Machida and Kuramochi1999), although with the absence of available supporting molecular data. Our multivariate cluster analysis showed distinct morphometrical convergence of the Japanese records of Machida and Kuramochi (Reference Machida and Kuramochi1999) with records of L. aniferum of Manter (Reference Manter1963) and that reported from off Ningaloo Reef (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023), coupled with divergence from records of S. acollum (see below). Comparative analysis of oral and ventral sucker widths of all specimens of L. aniferum distinctively separated Japanese records from those of Ningaloo Reef, the GBR, New Caledonia, and Fiji; however, Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) opted to identify their specimens as L. aniferum in the absence of any other supportive evidence (i.e., molecular data and/or distinction in the host). Species delimitation analyses of Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) revealed two distinct lineages in the Ningaloo Reef and Lizard Island specimens, separated by 22 bp in cox1 mtDNA and 1 bp in ITS2 rDNA sequences; 28S rDNA sequences were identical. Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) interpreted these molecular results as relating to geographical variation of a single species, possibly involving differences in overall size across different regions. Otherwise, results of supplementary species delimitation analyses by Mansour et al. (Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025) using available sequences of cox1 mtDNA for schistorchiines separated the Australian records of L. aniferum into two entities, despite being morphologically undifferentiated: specimens from off the Ningaloo Reef and those from off Lizard Island. Our multivariate cluster analysis is consistent with the conclusion of Mansour et al. (Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025), where specimens of L. aniferum from off the Ningaloo Reef were separate from those found off Lizard Island and within a distinct clade with specimens of Japan and Fiji (see below). According to the aforementioned, we interpret these data as consistent with the presence of cryptic species within Australian populations of L. aniferum made up of at least two groups: the Ningaloo Reef and the Lizard Island populations. Thus, L. aniferum (sensu lato) could represent a composite group of closely related species (i.e., cryptic species) not yet recognized/differentiated.
Lobatotrema droneni n. sp.
(Figures 3b, 4a–d)
Illustrations of Lobatotrema droneni n. sp. from the yellowmargin triggerfish, Pseudobalistes flavimarginatus (Rüppell). (a) Adult whole mount, paratype specimen, ventral view. (b) Terminal genitalia, dorsal view. (c) Proximal female genital system, dorsal view. (d) Elliptical eggs. Scale bars: (a) 500μm; (b, c) 200μm; (d) 100μm.
Taxonomic summary
Host: Yellowmargin triggerfish, Pseudobalistes flavimarginatus (Rüppell) (Balistidae).
Locality: Northern Red Sea, off Safaga, Southern Hurghada, Egypt.
Collection dates: 14/Mar/2019; 5/Oct/2019; 26/Dec/2020; 10/Jun/2022.
Site of infection: Intestine.
Prevalence: 2 out of 7 (28.6% infected).
Intensity: 8–11 worms.
Mean intensity: 9.5 (19/2) worms.
Relative density/abundance: 2.71 (19/7) worms.
LSID: 23B79EBB-88B0-469B-94B7-2DDDFA2ED599.
Deposited specimens: Parasitology Laboratory, Zoology Department, Faculty of Science, South Valley University; Collector YFMK, Holotype (SVU) 2025.19.Ld01; Paratypes (n = 4) (SVU) 2025.19.Ld02 & (SVU) 2025.22.Ld03– Ld05; Vouchers (n =10) (SVU) 2025.19. Ld06– Ld09 & (SVU) 2025.22.Ld10–Ld016.
Representative DNA sequences: ITS2 rDNA (accession no. PV567607) & 28S rDNA (accession no. PV567606).
Etymology: The species designation ‘droneni’ is named in honour and memory of Prof. Dr. Norman O. Dronen, Texas A & M University, and in recognition of his many contributions to our knowledge of the digenetic trematodes from the marine environment.
Description
[Based on 10 mature specimens]. Worms white in life. Body delicate, elongate to elongate linguiform with almost parallel margins; maximum width at equatorial region. Tegument thin, smooth. Anterior extremity nearly truncated to broadly rounded; posterior extremity slightly tapered, bluntly rounded. Forebody shorter than hindbody, slightly narrows near anterior end; hindbody larger, gradually widens near midbody then gradually narrows towards posterior extremity. Body superficially divided into two parts by a slight constriction at junction of first and second fifths of body. Eye-spot pigments few, dispersed in forebody. Pre-oral lobe short. Oral sucker elongate pyriform to elongate ovoid, highly glandular, ventro-subterminal, of ‘complex’ type, without circumoral lobes. Mouth conspicuous, directed antero-ventrally; surrounded by strong semi-circular muscle fibers that sometimes cover a large part of mouth aperture; partial V-shaped muscular sphincter half-encircles oral aperture postero-ventrally; elongated glandular cells surround mouth dorsally. Ventral sucker pre-equatorial, at end of anterior third of body, spherical, sessile, unspecialized, about one-third size of oral sucker. Suckers separated by short distance, about one-tenth of body length. Pre-pharynx absent. Pharynx subquadrate, immediately posterior to or slightly overlapped by oral sucker, separated from ventral sucker by a short distance, weakly developed, not papillate, slightly smaller than ventral sucker. Oesophagus not evident. Intestinal bifurcation near end of forebody, immediately posterior to pharynx, separated from ventral sucker by a short distance. Caeca two, simple, slightly wider anteriorly, slightly arcuate posteriorly, equal in length, extend parallel along lateral margins; terminate at posterior extremity in small, separate ani; anus marginal, narrow, subterminal.
Testes two, smooth, sub-elliptical to globular, subequal in size, contiguous, occupies majority of second third of body or its posterior half. Post-testicular space occupies last third of body. Seminal vesicle saccate with narrower distal portion, large, median, sub-elliptical, unipartite, median to slightly dextral, extends well into hindbody, vertically overlaps anterior half or more of ovary and ventral sucker dorsally, terminates immediately posterior to intestinal bifurcation. Pars prostatica tubular, short, thin-walled, overlaps anterior margin of ventral sucker, forms very short hermaphroditic duct; duct itself cylindrical, very small, extends antero-dorsally to ventral sucker. Prostatic cells few, free in parenchyma, surrounds pars prostatica and hermaphroditic duct. Genital pore median, immediately post-bifurcal.
Ovary oval, smooth, in posterior portion of anterior third of body, dextro-submedian, contiguous to posterior margin of ventral sucker, either entirely pre-testicular or sometimes overlaps anterior margin of anterior testis ventrally. Canalicular seminal receptacle conspicuous, saccate, size of ovary or slightly smaller, para-ovarian, sinistro-submedian. Right and left transverse vitelline ducts unite posterior to ventral sucker; small median vitelline reservoir opens into oötype. Vitellarium follicular; extends from end of pharynx or just anterior to ventral sucker along intestinal caeca to close to posterior extremity; mainly restricted to lateral margins anteriorly, becomes more extensive posteriorly and occupies most available space around and posterior to testes; overlaps lateral and posterior margins of testes, dorsally non-confluent immediately posterior to testes, then broadly separated into four poorly defined columns of follicles, which are themselves numerous, small, sub-spherical to oval, lateral, dorsal, and ventral to caeca. Uterus para-ovarian, short, saccular, intercaecal, restricted to area between ovary, seminal receptacle, seminal vesicle, left cecum and genital pore; dorsally overlaps left edge and extends antero-sinistrally around left side of ventral sucker aperture into metraterm. Post-uterine distance very long, about last two-thirds of body. Metraterm tubular, short, wide, runs short distance parallel to left and anterior margins of ventral sucker, opens into hermaphroditic duct at post-bifurcal level. Eggs few (approx. 4–23), operculated, oval, thin-shelled, non-filamented.
Excretory vesicle I-shaped, median, wider anteriorly, extends to near level of posterior testis. Excretory pore terminal.
Variability in adult specimens
(Table 3)
Measurements, morphometric percentages, and morphometric ratios of Lobatotrema droneni n. sp. compared to Lobatotrema aniferum Manter, Reference Manter1963 (sensu lato)
--- = Neither given in original description nor available from published illustration(s).
[ ] Values outside the original description as a result of either the potential inaccuracy of original calculations and/or values of scale bars.
() Mean values of the target measurement.
a Yellowmargin triggerfish, Pseudobalistes flavimarginatus (Rüppell).
b Proportion of body length; range values are given first followed by the mean values.
c Calculated from measurements given in the original description.
d Calculated from the illustrations of the original description (Manter Reference Manter1963, Figure 8).
e CV, coefficient of variation; r, Pearson correlation coefficient; P, probability value.
Measurements of oral sucker length and ratios of oral sucker length, pre-ovarian distance, and pre-testicular distance relative to body length exhibit low CV of a strong significant correlation with body length (r≥0.78, p<0.05). Other metric characters exhibit low CV with non-significant correlation to the body length (r≤0.22, p>0.05), incorporating measurements of pre-genital pore distance and egg length in addition to body width and post-uterine distance as percentages of body length. Measurements of pre-ovarian distance and the ratio of oral sucker width relative to body length show a moderate to strong correlation with body length (r≥0.55), though not statistically significant (p>0.05). Out of 22 morphometric characters with moderate CV, measurements of body width, oral sucker width, and pre-bifurcal distance in addition to ratios of ventral sucker width and pre-genital pore distance as % of body length and the pharynx/oral sucker length ratio show a significant correlation with body length (r≥0.68, p<0.05). Regarding metric characters with high CV, measurements and ratios of distance from ventral sucker to ovary and inter-testicular space are notably the largest (CV>183). Sixteen metric characters of high CV exhibit a significant correlation with body length (r≥0.68, p≤0.05), incorporating measurements of ovary length, seminal receptacle length, testes size, post-testicular distance, post-uterine distance, and distance from ventral sucker to intestinal bifurcation and genital pore along with percentages of ventral sucker size, seminal vesicle size, pre-bifurcal distance, pre-genital pore distance, pre-vitelline distance relative to body length, and the ratio of pharynx/ventral sucker length.
Remarks
This second digenean species also belongs within the Schistorchiinae based on overall morphological diagnostic features and a marine fish host (Blend et al. Reference Blend, Karar and Dronen2017; Cribb Reference Cribb, Jones, Bray and Gibson2005; Cribb and Bray Reference Cribb and Bray1999; Pulis et al. Reference Pulis, Curran, Andres and Overstreet2014). Our material is placed within Lobatotrema based on the following combination of morphological and ecological diagnostic features: two testes, caeca open independently via separate ani, parasitizing species of Pseudobalistes (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023), and a V-shaped oral sphincter and dextro-submedian ovary. With the unique combination of morphological features, supported by morphometric percentages and ratios (see below; Tables 3, S1) and distinctive molecular data, our specimens are identified as a new species of Lobatotrema.
Lobatotrema droneni n. sp. differs from L. aniferum (sensu lato) in its (i) large seminal vesicle that runs longitudinally, extending well into the hindbody and medially overlapping the anterior half of the ovary and ventral sucker dorsally to terminate immediately posterior to the intestinal bifurcation vs a small, transversely oriented, dextral seminal vesicle, terminating immediately at the anterior end of the ventral sucker; (ii) post-bifurcal genital pore vs a genital pore situated immediately anterior to the ventral sucker; (iii) para-ovarian and sinistro-submedian seminal receptacle the size of the ovary or slightly smaller vs a seminal receptacle positioned dorsally to ovary; and (iv) superficial division of the body plan into two parts via a slight constriction at the end level of the oral sucker in contrast to a uniform body shape in L. aniferum. Measurements of L. droneni n. sp. are smaller, generally, than those of L. aniferum (sensu lato) (see Table 3; Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023, Table 4). Although several allometric percentages and ratios of L. aniferum (sensu lato) fall within the range of L. droneni n. sp., the latter is characterized by substantially smaller body size; a smaller body width ratio of about a quarter of body length vs ratios exceeding a third of body length; a larger oral sucker about one-fifth of body length with a higher value of pharynx length ratio relative to body length compared to lower values in L. aniferum; and a post-testicular distance of about the last third of the body compared to a larger distance exceeding two-fifths of body length in L. aniferum. Lobatotrema droneni n. sp. is differentiated from L. aniferum ex. P. flavimarginatus by a lower pharynx/ventral sucker length ratio; a larger pre-testicular space of mean length exceeding two-fifths of body length vs values around a third of body length; a longer pre-ovarian distance of mean length exceeding two-fifths of body length compared to values less than one-third of body length; and slightly smaller egg sizes (see Table 3). Supporting morphometrical results, our multivariate cluster analysis suggests separation of our specimens into their own cluster away from other records of L. aniferum (sensu lato) and S. acollum (see below; Table S1). Regarding molecular data, ITS2 rDNA and 28S rDNA sequence data for L. droneni n. sp. and L. aniferum differ at 2 and 3 bp, respectively. In the phylogenetic analysis, the two species are gathered in a strongly supported clade, in which L. droneni n. sp. is a sister/basal clade to L. aniferum (sensu lato).
Host-parasite data show that P. flavimarginatus is widely distributed from the Red Sea to East Africa, to northern Australia, the Tuamotu Islands, and southern Japan (Froese and Pauly Reference Froese and Pauly2025). However, the Red Sea is a more distant and enclosed sea of warmer, saltier water with unique marine life and distinct ecosystems compared to those of other Indo-Pacific regions (Castro and Huber Reference Castro and Huber2010; Stewart Reference Stewart2008; Tesfamichael and Pauly Reference Tesfamichael and Pauly2016). Thus, the possibility of a variety of associated/closely related digeneans parasitizing the same host species is plausible. For instance, Bray and Cribb (Reference Bray and Cribb1998) described Pelopscreadium spongiosum (Bray & Cribb, Reference Bray and Cribb1998) Dronen, Blend, Khalifa, Mohamadain & Karar, Reference Dronen, Blend, Khalifa, Mohamadain and Karar2016 (syn. Bianium spongiosum Bray & Cribb, Reference Bray and Cribb1998) (Lepocreadiidae) off Lizard Island, Queensland, Australia from the yellow boxfish, Ostracion cubicus Linnaeus (Tetraodontiformes: Ostraciidae). From the same host species, Dronen et al. (Reference Dronen, Blend, Khalifa, Mohamadain and Karar2016) characterized Pelopscreadium aegyptense Dronen, Blend, Khalifa, Mohamadain & Karar, Reference Dronen, Blend, Khalifa, Mohamadain and Karar2016 from the Red Sea off Sharm El-Naga, Egypt. Similarly, Japanese and Ningaloo Reef records of L. aniferum (from B. viridescens and P. fuscus) represent a distinct group from those of New Caledonian and Lizard Island records from the same hosts, despite being indistinguishable morphologically (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023). Furthermore, Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) described two species of Blendiella from the same hosts, the orange-lined triggerfish, Balistapus undulatus (Park), and the halfmoon triggerfish, Sufflamen chrysopterum (Bloch & Schneider) (Tetraodontiformes: Balistidae), within the same locality (off Lizard Island, Australia).
According to the distinctive morphology of body outline and genitalia, significant morphometric variation, molecular distinction, multivariate cluster analysis, and comparatively distant geographic locality (Northern Red Sea), and in light of criteria proposed by Bray et al. (Reference Bray, Cutmore and Cribb2022) for trematode species recognition, we consider these differences evidence for considering L. droneni n. sp. a distinct species; however, one similar to populations of L. aniferum (sensu lato).To our knowledge, we did not find any record of Lobatotrema spp. from the Red Sea, so the finding of L. droneni n. sp. herein widens the distribution range of this genus.
Molecular analysis
Genetic distances
Three datasets were constructed using the ITS2 rDNA, partial 28S rDNA, and a combined 28S rDNA+ITS2 rDNA. The 28S rDNA region was aligned with 56 sequences representing all available and relevant megaperid taxa: 35 sequences from four species representing the Apocreadiinae, four sequences from three species representing the Megaperinae, and 15 sequences from five species representing the Schistorchiinae with four sequences representing three species from the Haploporidae Nicoll, 1914 for outgroup comparisons. The final 28S rDNA alignment comprised 58 sequences, totalling 1,265 base pairs. Of these, 781 (61.8%) were conserved, 483 (38.2%) were variable, 388 (30.7%) were parsimony-informative, and 95 (7.5%) were singleton sites. The genetic divergence within megaperids ranged from 0% to 13.2%. The ITS2 rDNA comparisons included 60 taxa of megaperids: 33 sequences from four species representing the Apocreadiinae, four sequences from three species representing the Megaperinae, and 23 sequences representing 5 species from the Schistorchiinae with three haploporid sequences for outgroup comparisons. The final alignment ITS2 rDNA datasets comprised 63 sequences, yielding a total of 425 aligned positions, with 210 (51.1%) bp conserved sites and 208 bp (48.9%) variables. Of these, 176 bp (41.4%) were parsimony informative sites, 30 bp (7.1%) were singleton sites, and a genetic divergence range of 0%–19.1% within megaperids. The combined 28S rDNA+ITS2 rDNA comparisons comprised 54 taxa of the Megaperidae: 35 sequences from four species representing apocreadiines, four sequences from three species of megaperines, and 15 sequences from five species of schistorchiines with three haploporid sequences for the outgroup comparisons. The final alignment of the combined 28S rDNA+ITS2 rDNA datasets included 57 sequences, resulting in a 1,678 bp alignment, comprising 991 (58.8%) bp conserved sites and 691 bp (41.2%) variables, with 564 bp (33.4%) parsimony-informative sites, 125 bp (7.4%) singleton sites, and a genetic divergence range of 0%–15.1% within megaperids.
Sequences of L. aniferum from Ningaloo Reef and that from off Lizard Island are identical, with no nucleotide differences in the 1,265 bp long 28S rDNA dataset, whereas they differ by just 1 bp (0.2%) in the 425 bp long ITS2 rDNA dataset. Lobatotrema droneni n. sp. exhibits a variation of 2–3 bp (0.5%–0.7%) and 2 bp (0.2%) in the ITS2 rDNA and the 28S rDNA datasets, respectively, compared to the two Australian forms of L. aniferum. ITS2 rDNA and 28S rDNA sequence data for S. acollum differ from those of L. aniferum at 15–16 (3.5%–3.8%) and 64 (5.1%) bp, respectively, and from L. droneni n. sp. at 14 (3.3%) and 63 (5.0%) bp, respectively.
Phylogenetic analysis
Bayesian phylogenetic tree illustrating phylogenetic relationships among taxa of the Megaperidae, Manter, Reference Manter1934, inferred from Maximum likelihood (ML) and Bayesian inference (BI) analyses of the 28S rDNA dataset. Solid circles represent nodal support with posterior probabilities for the BI analysis ≥ 0.95 and bootstrap scores for the ML analysis ≥ 70%. Branch length refers to the number of substitutions per site. Scale bar indicates the expected number of substitutions per site.
Bayesian phylogenetic tree illustrating phylogenetic relationships among taxa of the Megaperidae, Manter, Reference Manter1934, inferred from Maximum likelihood (ML) and Bayesian inference (BI) analyses of the ITS2 rDNA dataset. Solid circles represent nodal support with posterior probabilities for the BI analysis ≥ 0.95 and bootstrap scores for the ML analysis ≥ 70%. Branch length refers to the number of substitutions per site. Scale bar indicates the expected number of substitutions per site.
Bayesian phylogenetic tree illustrating phylogenetic relationships among taxa of the Megaperidae, Manter, Reference Manter1934, inferred from Maximum likelihood (ML) and Bayesian inference (BI) analyses of the combined 28S rDNA+ITS2 rDNA datasets. Solid circles represent nodal support with posterior probabilities for the BI analysis ≥ 0.95 and bootstrap scores for the ML analysis ≥ 70%. Branch length refers to the number of substitutions per site. Scale bar indicates the expected number of substitutions per site.
Phylogenetic analyses of the three datasets resulted in the ingroup taxa of the Megaperidae forming a monophyletic clade to the exclusion of the outgroup taxa (Figures 5–7). Three subfamilies are recognized, each resolved as a highly supported monophyletic clade (BI=1; ML≥95): the Schistorchiinae, the Megaperinae, and the Apocreadiinae. The 28S rDNA and 28S rDNA+ITS2 rDNA phylogenetic trees resolved schistorchiines and megaperines as sister clades forming a strongly supported Schistorchiinae/Megaperinae clade (1/100), which in turn, resolved as a sister to the Apocreadiinae (Figures 5 and 7). The ITS2 rDNA phylogenetic tree depicted the Megaperidae as a 3-way polytomy, each branch corresponded to a distinct subfamily (Figure 6).
28S rDNA differentiated taxa within the Schistorchiinae into two sister clusters: a weakly supported Lobatotrema/Paraschistorchis Blend, Karar & Dronen, Reference Blend, Karar and Dronen2017 clade (0.82/66) and a strongly supported larger clade (1/92) constituting Sphincteristomum, Schistorchis (sensu stricto) Lühe, 1906, and Blendiella Magro, Cutmore, Carrasson & Cribb, Reference Magro, Cutmore, Carrasson and Cribb2023. Similarly, the ITS2 rDNA tree divided schistorchiines into two sister clades: a clade of Paraschistorchis spp. only with a slightly weak support value (0.92/65) and a large cluster of remaining schistorchiines (Lobatotrema, Schistorchis [sensu stricto], Sphincteristomum, and Blendiella) with high support (1/81). Likewise, the combined 28S rDNA+ITS2 rDNA tree exhibited the same previous division patterns of schistorchiines as in the ITS2 rDNA tree, however, with clearly resolved relationships and high support values among all their members (BI=1; ML≥91).
Sphincteristomum acollum exhibited a constant phylogenetic position of high support (BI≥0.99; ML≥70) as a sister to Blendiella spp. in all three trees. In contrast, the position of Lobatotrema spp. fluctuated among the three phylogenetic trees from being a sister to taxa of Paraschistorchis in the 28S rDNA tree, or sister to the Sphincteristomum/Blendiella clade in the ITS2 rDNA tree, to being completely resolved in a basal position with high support to a group composed of Schistorchis, Sphincteristomum, and Blendiella in the combined 28S rDNA+ITS2 rDNA tree. Members of Lobatotrema are resolved as a monophyletic clade of high support in all three phylogenetic trees, with L. droneni n. sp. sister/basal to L. aniferum (sensu lato).
Remarks
We have used the results of the combined 28S rDNA+ITS2 rDNA tree to build our observations and discussions rather than the separate 28S rDNA and ITS2 rDNA trees, as the former is more informative and gives fully resolved relationships using the same taxa of the Schistorchiinae that are used for constructing the latter two trees.
Four main features have been used to distinguish the eight genera of the Schistorchiinae we recognize (see WoRMS 2025), including the number of testes (two or multiple), the terminal nature of the caeca (ending in ani, uroproct, or blindly), oral sucker morphology (‘complex’ or ‘simple’), and distinction of the host group (Blend et al. Reference Blend, Karar and Dronen2017; Cribb Reference Cribb, Jones, Bray and Gibson2005; Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023; Mansour et al. Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025).
Blend et al. (Reference Blend, Karar and Dronen2017) extensively addressed oral sucker morphology of schistorchiines and concluded its differentiation into a ‘complex’ and a ‘simple’ nature. The ‘complex’ nature of the oral sucker is characterized by a large size relative to body size, being ≥ 2 times the size of the ventral sucker, the presence of circumoral lobes, and a predominantly glandular appearance. The oral sucker of a ‘simple’ nature is characterized by being smaller in size than that of the ‘complex’ nature, lacking circumoral lobes and being entirely muscular (see Blend et al. Reference Blend, Karar and Dronen2017). Our phylogenetic analysis exhibits division of schistorchiines into two highly supported clusters: a clade of Paraschistorchis spp. and a clade comprising Lobatotrema, Schistorchis, Sphincteristomum [sensu stricto], and Blendiella (Figure 7). The most distinguishing synapomorphy among the latter four genera is the ‘complex’ nature of the oral sucker compared to an oral sucker of a ‘simple’ nature in Paraschistorchis. Thus, our phylogenetic results follow a pattern of internal classification of schistorchiines based on differences in oral sucker morphology. Mansour et al. (Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025) referred to such a result, in part, upon referring to a notable phylogenetic divergence between taxa of Schistorchis and Paraschistorchis compared to convergence among Schistorchis, Sphincteristomum and Blendiella. By extension, schistorchiines of ‘complex’ oral sucker nature (i.e., Blendiella, Lobatotrema, Schistorchis, and Sphincteristomum [sensu stricto]) are expected to be phylogenetically grouped in their own clade compared to clustering schistorchiines of ‘simple’ oral sucker nature (i.e., Neomegacreadium Machida & Kuramochi, Reference Machida and Kuramochi1999, Paraschistorchis, Plesioschistorchis Blend, Karar & Dronen, Reference Blend, Karar and Dronen2017, and Sphincterostoma Yamaguti, Reference Yamaguti1937) without confusion or overlap between members of the two groups. Hence, our results match the scheme proposed by Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) regarding the division of the eight genera of the Schistorchiinae into two main phylogenetic groups according to the nature of the oral sucker (i.e., glandular / ‘complex’ or muscular / ‘simple’).
Within the clade of schistorchiines having an oral sucker of ‘complex’ nature, we find that the taxa of Lobatotrema are completely resolved in a basal position to the other genera and distinctly phylogenetically separated from Sphincteristomum (Figures 5–7). Besides confirming the validity of the concept of Lobatotrema from Sphincteristomum (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023; Mansour et al. Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025, Figure 4), our analysis reveals another pattern of internal classification based on the shape of the oral sphincter. The most distinguishing synapomorphy among Blendiella, Schistorchis (sensu stricto), and Sphincteristomum is the U-shaped oral sphincter in contrast to a V-shaped oral sphincter in Lobatotrema. Regarding oral sucker shape within schistorchiines, each genus possesses a distinct shape, either evenly round in outline to subglobular or pyriform. However, it is not reflected in our phylogenetic tree (Figure 7). Species of Blendiella and Lobatotrema are phylogenetically separated despite having a pyriform oral sucker. Likewise, Sphincteristomum and Schistorchis are also phylogenetically distant despite possessing a rounded oral sucker. Still, oral sucker shape can be used as an additional morphological feature to differentiate between closely related genera. For instance, Blendiella spp. possess a pyriform oral sucker, whereas those of species of Schistorchis (sensu stricto) are rounded (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023). Through our investigation and review of schistorchiines having a ‘complex’ oral sucker, we find using the combination of the shape of the oral sucker and its oral sphincter represents a more effective differential feature between schistorchiine genera, in general, and those more closely related, in particular. For instance, Lobatotrema spp. possess an ovoid to pyriform oral sucker with a V-shaped oral sphincter compared to a round to subglobular oral sucker with a U-shaped oral sphincter in Sphincteristomum spp., which in turn, may explain their distinct phylogenetic separation.
Although the number of testes (either two or multiple) represents one of the main differential characteristics that keys recognize and use to separate genera of the Schistorchiinae into two main groups (Blend et al. Reference Blend, Karar and Dronen2017; Cribb Reference Cribb, Jones, Bray and Gibson2005; Cribb and Bray Reference Cribb and Bray1999; Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023), this feature is not reflected in our phylogenetic tree in the clade of schistorchiines having an oral sucker of ‘complex’ nature. Blendiella spp. and Schistorchis spp. do not form a monophyletic clade, although both have numerous testes and are morphologically similar (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023), whereas the former constitutes a strongly supported clade with Sphincteristomum that possesses two testes and is distinctly morphologically different. Likewise, Lobatotrema forms a clade distant from Sphincteristomum despite both having two testes and being closely related morphologically. Such a close phylogenetic relationship between Blendiella and Sphincteristomum is likely subject to the close ecological/phylogenetic relationship between their corresponding hosts. Species of Blendiella are recorded from balistid fishes within Balistapus Tilesius, Sufflamen, and Rhinecanthus. In addition to the latter two fish genera, Sphincteristomum spp. are recorded from Abalistes, too. All four fish genera are phylogenetically close and distributed in two strongly supported sister clades (McCord and Westneat Reference McCord and Westneat2016, Figure 1, clades 2,3). Species of Schistorchis are recorded from monacanthid, tetraodontid, and triacanthid fishes within the Tetraodontiformes (see Blend et al. Reference Blend, Karar and Dronen2017; Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023), a group of fishes distinctly different ecologically/phylogenetically from balistid fishes, which in turn, can explain, at least in part, the phylogenetic divergence between species of Schistorchis and those of Blendiella.
Regarding the distinction of the host group(s) in the Schistorchiinae, our phylogenetic analysis, coupled with a comprehensive review of records of schistorchiines, suggests almost all recognized schistorchiine species/genera are oioxenic (i.e., restricted to a specific host species) or stenoxenic parasites (i.e., parasitize a narrow range of phylogenetically/taxonomically associated hosts) (Figures 5–7; cf. Mansour et al. Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025). Such results follow distribution and specificity patterns of several host-parasite studies of the Schistorchiinae (see Blend et al. Reference Blend, Karar and Dronen2017; Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023; Mansour et al. Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025) except for some disputed taxa—in particular, L. aniferum (sensu lato) and S. carneus (sensu lato) (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023; Mansour et al. Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025; Present study). Another observation is that the present phylogenetic results exhibit indeterminism in grouping schistorchiine genera into a single clade based on their hosts belonging to the same family. For example, taxa of Lobatotrema (sensu stricto) and Sphincteristomum are known from balistid fishes; some of these fish species are environmentally associated and phylogenetically closely related (see McCord and Westneat, Reference McCord and Westneat2016, Figure 1, clades 1–3). However, no convergence between both genera is reflected in our phylogenetic tree – alternatively, they are phylogenetically distant (see Figures 5–7). Accordingly, we find that (i) variation in the host group does not clearly reflect on the phylogenetic analysis of schistorchiine genera as does oral sucker morphology; (ii) the distinction of host group works to differentiate among parasite genera and across their representative species, even those morphologically difficult to distinguish, such as Lobatotrema and Sphincteristomum (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023; Present study), as well as for potential species complexes in the stenoxenic L. aniferum (sensu lato) and S. carneus (sensu lato) (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023; Mansour et al. Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025; Present study); (iii) in some cases, the same host species is reported harboring two schistorchiine species. Typically, both taxa belong to the same genus and are very similar morphologically with only narrow differences in appearance. For instance, Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) recorded two stenoxenic Blendiella spp. from the same hosts (B. undulatus and S. chrysopterum) within nearby localities off Lizard Island, Australia. From the same host, P. flavimarginatus, within two distant localities, we reported L. droneni n. sp. from off Safaga, Egypt, Northern Red Sea, whereas Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) recorded L. aniferum (sensu lato) from off Lizard Island, Australia. In another instance, species delimitation analyses of schistorchiines by Mansour et al. (Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025) were consistent with the presence of two entities within the Australian populations of Paraschistorchis stenosoma despite being from the same host (the honeycomb filefish, Cantherhines pardalis [Rüppell] [Tetraodontiformes: Monacanthidae]) at nearby localities (Lizard Island vs Heron Island). In light of these remarks, our findings suggest that it may be common to find morphologically closely related schistorchiine species within the same host, either collected from distant or nearby localities.
Cluster analysis
Dendrogram illustrating the hierarchical clustering of analyzed records of Lobatotrema droneni n. sp. with those calculated from original descriptions/redescriptions of Lobatotrema aniferum Manter, Reference Manter1963 (sensu lato) and Sphincteristomum acollum Oshmarin, Mamaev & Parukhin, Reference Oshmarin, Mamaev and Parukhin1961, generated using Ward’s method and squared Euclidean distance. Abbreviations: ave, average recorded values of the target taxon; max, maximum recorded values of the target taxon; min, minimum recorded values of the target taxon; R1, record of L. aniferum ex. Balistoides viridescens (Bloch & Schneider) off Lizard Island; R2, record of L. aniferum ex. Pseudobalistes flavimarginatus (Rüppell) off Lizard Island; R3, record of L. aniferum ex. Pseudobalistes fuscus (Bloch & Schneider) off New Caledonia; R4, record of L. aniferum ex. P. fuscus off Ningaloo Reef; R5, record of S. acollum ex. Abalistes stellatus (Anonymous) off Lizard Island; Sa, sample.
The resultant dendrogram of hierarchical agglomerative clustering (HAC) divides target records of S. acollum, L. aniferum (sensu lato), and L. droneni n. sp. into two groups; the larger one divides into two sister clusters. Thus, three main clusters are observed and are arranged from the closest to the most distant record. Cluster (A) consists of records of S. acollum from the Gulf of Tonkin (Oshmarin et al. Reference Oshmarin, Mamaev and Parukhin1961) and off Lizard Island (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023) as well as records of L. aniferum from Ningaloo Reef (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023), off Okinawa, Japan (Machida and Kuramochi Reference Machida and Kuramochi1999) and Fiji (Manter Reference Manter1963). Cluster (B) comprises records of L. aniferum ex. B. viridescens and P. flavimarginatus off Lizard Island, and L. aniferum ex. P. fuscus off New Caledonia (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023), in addition to one specimen of L. droneni n. sp. Cluster (C) contains all records of L. droneni n. sp. except for one specimen. The HAC analysis shows that i) Cluster (A) and Cluster (B) are the most convergent groups, whereas Cluster (C) is the most distant in a basal position to the former clusters; ii) within Cluster (A), specimens of S. acollum are positioned basal to records of L. aniferum from Ningaloo Reef, off Okinawa and Fiji; iii) within Cluster (B), the three records of Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) show an overlap with each other, which reflects their high morphometric convergence; and iv) specimens of S. acollum represent an intermediate barrier that separated records of L. aniferum collected off Lizard Island and New Caledonia from those from Ningaloo Reef and off Okinawa and Fiji.
Remarks
Positioning of specimens of L. droneni n. sp. in their own cluster away from other records of S. acollum and L. aniferum (sensu lato) is attributed to a combination of some distinctive allometric percentages and ratios compared to other records. These combinations include smaller values in body width, anterior testis width, and post-testicular distance as well as larger values in forebody length, oral sucker length, distance from ventral sucker to both intestinal bifurcation and genital pore, anterior testis length, egg length, egg width, pharynx length/oral sucker length ratio, pharynx length/ventral sucker length ratio, ovary length/width ratio and anterior testes length/width ratio (see Table S1). These metric differences are compatible with the previously mentioned distinctive morphological features and phylogenetic analysis of our specimens of L. droneni n. sp. When compared to L. aniferum (sensu lato) and its closely related S. acollum (see Results above), such metric differences present additional supportive evidence that our specimens represent a new taxon within Lobatotrema. Regarding the divergence of one specimen of L. droneni n. sp. from Cluster (C) into Cluster (B), this specimen is in a basal position to all its records and is clearly well-separated from as well as mediates between Cluster (C) and other remaining records of Cluster (B). This reflects a difference of this specimen from those in Cluster (B), and the convergence can be attributed to the fact that this specimen exhibits morphometrical convergence with those of Cluster (B) rather than those of Cluster (A). The presence of a few outliers among several morphometric measurements of the target taxon is common and acceptable in nature.
Insertion of records of S. acollum between records of L. aniferum (sensu lato) shows close morphometric convergence, which in turn, is compatible with the highly close morphology between the two species. Such close morphology/morphometry caused an overlap and confusion between S. acollum and L. aniferum. Hence, it was one of the main reasons for considering that both belong to the same genus (i.e., Sphincteristomum) and species (i.e., S. acollum) (see Blend et al. Reference Blend, Karar and Dronen2017; Cribb Reference Cribb, Jones, Bray and Gibson2005; Cribb and Bray Reference Cribb and Bray1999) until recently (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023; Present study). Our investigations around records of S. acollum reveal a combination of some distinctive allometric percentages and ratios that should be carefully considered in comparison with L. aniferum (sensu lato), and which may represent diagnostic and discriminatory characteristics between the two species. These metric characteristics include the highest values in body width (greater than 50% body length), oral sucker width, body width/forebody length ratio, and oral sucker length/oral sucker width ratio as well as lower values in pre-vitelline distance, distance between suckers, pharynx width/oral sucker width ratio, and ventral sucker/oral sucker ratios (see Table S1). The record of S. acollum from the Gulf of Tonkin (Oshmarin et al. Reference Oshmarin, Mamaev and Parukhin1961) is resolved as basal to that from off Lizard Island, Australia (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023), which in turn is basal to the group of records of L. aniferum from Japanese, Ningaloo Reef, and Fiji waters. Non-clustering of records of S. acollum in one clade can be explained in that measurements of the Australian record of S. acollum were reported from an incomplete single hologenophore missing part of its posterior end (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023); thus, its related measurements were affected. In contrast, almost all other morphometric measurements are strongly consistent with the original species description (see Table S1).
The morphometric divergence between the two groups of L. aniferum (sensu lato) (i.e., a population of records within Cluster [A] and another population within Cluster [B]) is consistent with the presence of two morphologically undifferentiated entities within records of L. aniferum (sensu lato); hence, this could at least represent a repository of two closely related cryptic species, neither recognized yet. This result is compatible with (i) molecular results of Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) who inferred that specimens of L. aniferum from Ningaloo Reef and Lizard Island formed two distinct lineages, differing by 22 base pairs (bp) in cox1 mtDNA sequences and one bp in ITS2 rDNA, as well as (ii) supplementary species delimitation analyses of cox1 mtDNA by Mansour et al. (Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025) who split records of L. aniferum into two entities, one from Ningaloo Reef and one from Lizard Island, despite their morphological similarity. Comparative analysis of oral and ventral sucker widths of specimens of L. aniferum (sensu lato) suggested two divergent groups of specimens: Japanese records versus records of Ningaloo Reef, the GBR, New Caledonia, and Fiji (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023). On the contrary, present HAC clarifies the close similarity between Japanese records and those of Ningaloo Reef and Fiji, whereas records of Lizard Island and New Caledonia are the most metrically dissimilar (see Figure 8). Thus, records of L. aniferum (sensu lato) apparently show a species complex that can be divided at least into two population compositions, metrically divergent but indistinguishable morphologically: a group that includes Japanese records, the Ningaloo Reef population, and Fiji records vs a group of Lizard Island and New Caledonia populations (see Figure 8). Differences between the two groups can be attributed to a combination of relative divergence in some morphometric percentages and ratios. Japanese records and those of Ningaloo Reef and Fiji show higher values in anterior and posterior testis width as well as lower values in egg length, egg width, pharynx length/ventral sucker length ratio, mean testes length/ovary length ratio, and ovary length/ovary width ratio (see Table S1).
Discussion
Problematic taxonomic status of Sphincteristomum mediterraneae Abid-Kachour, Mouffok & Boutiba, Reference Abid-Kachour, Mouffok and Boutiba2013
Illustrations of Sphincteristomum mediterraneae Abid-Kachour, Mouffok & Boutiba, Reference Abid-Kachour, Mouffok and Boutiba2013 (species inquirendum) from the common pandora, Pagellus erythrinus (Linnaeus). (a) Immature specimen of S. mediterraneae modified after Abid-Kachour et al. (Reference Abid-Kachour, Mouffok and Boutiba2013, Figure 3a,b), ventral view. (b) Ovigerous specimen of S. mediterraneae modified after Abid-Kachour et al. (Reference Abid-Kachour, Mouffok and Boutiba2013, Figure 4a,b), ventral view. Scale bars: (a, b) 200μm.
Sphincteristomum mediterraneae falls within the concept of the Schistorchiinae based on having an oral sucker with a partial U-shaped sphincter half encircling the oral aperture postero-ventrally combined with post-ovarian testes and an ovary in the mid hindbody (see Blend et al. Reference Blend, Karar and Dronen2017; Cribb Reference Cribb, Jones, Bray and Gibson2005). However, our comprehensive revision and detailed comparisons suggest an unambiguous distinction of S. mediterraneae from other species of Sphincteristomum (sensu lato) and a controversial and more ambiguous taxonomic status at the subfamily level (see below).
Sphincteristomum mediterraneae exhibits a unique combination of several differential morphological characteristics compared to all schistorchiines having two testes (Lobatotrema, Sphincterostoma Yamaguti, Reference Yamaguti1937 and the other taxa of Sphincteristomum [sensu stricto]): (i) an inner, highly muscular, lobed oral sphincter surrounded by circular and longitudinal muscles and numerous gland-like cells; (ii) a wavy-edged pharynx vs a smooth one; (iii) two caeca that terminate at the level of the anterior end of the excretory vesicle/second third of the body (see Abid-Kachour et al. Reference Abid-Kachour, Mouffok and Boutiba2013, Figures 2b, 3a,b, 4a–c) in contrast to caeca terminating at the posterior extremity of the body; (iv) extracaecal vitelline follicles arranged into two narrow bands that converge in the hindbody around the lateral outer edges of the excretory vesicle (see Abid-Kachour et al. Reference Abid-Kachour, Mouffok and Boutiba2013, Figure 4b,c) compared to a large vitelline field, confluent in the post-testicular region with profuse follicles that are distributed dorsal, lateral, and ventral to caeca; (v) a sinuous/coiled seminal vesicle vs a saccate one; (vi) the termination of distal portions of the male and female ducts into the genital atrium separately without forming a hermaphroditic duct (see Abid-Kachour et al. Reference Abid-Kachour, Mouffok and Boutiba2013, Figures 3b,c, 4a,c); (vii) the smallest post-testicular region and the shortest excretory vesicle, about one-quarter of body length or less; (viii) the smallest egg (30 × 24), about half the reported sizes; and (ix) S. mediterraneae has a distinct host group and a considerably distant/distinct locality (sparid fishes in the Eastern Atlantic Ocean [Mediterranean Sea] vs balistid, silurid and malacanthid fishes in the Indo-Pacific Ocean). Compared to Lobatotrema spp. and the other taxa of Sphincteristomum (sensu stricto), we find S. mediterraneae is also characterized by its highly muscular oral sucker and an ovary separated from the ventral sucker by uterine coils in contrast to a highly glandular oral sucker and an ovary continuous with the ventral sucker. Morphometrically, S. mediterraneae has the smallest body size and, by extension, dimensions of internal organs; however, it exhibits the highest values in several morphometric percentages/ratios compared to that of Lobatotrema spp. and other taxa of Sphincteristomum (sensu stricto) as follows: oral sucker length, ventral sucker length, distance between suckers, distance from ventral sucker to ovary, distance separating ventral sucker and testes, and the seminal vesicle, pre-vitelline, pre-ovarian, and pre-testicular lengths as percentages of body length (see Table 2). Additionally, S. mediterraneae is distinguished from Lobatotrema spp. by its ovate body, longer forebody, spherical to subspherical oral sucker with a U-shaped sphincter, median genital pore, and median ovary. Although Sphincterostoma spp. have a muscular oral sucker and an unarmed tegument as in S. mediterraneae, the former has an elongated body and a submedian ovary as well as caeca that unite in the posterior region of the hindbody to form a uroproct (see Cribb Reference Cribb, Jones, Bray and Gibson2005; Yamaguti Reference Yamaguti1937; Zaidi and Khan Reference Zaidi and Khan1977).
Concerning the Megaperinae Manter, Reference Manter1934, S. mediterraneae share with members of this subfamily some distinctive features, in particular, a papillate ‘waved-edged’ pharynx, a short genital atrium, and a host distributed in the Atlantic Ocean (see Bray Reference Bray, Jones, Bray and Gibson2005b; Cribb Reference Cribb, Jones, Bray and Gibson2005; Pulis et al. Reference Pulis, Curran, Andres and Overstreet2014). Sphincteristomum mediterraneae can be differentiated from known megaperines by its smooth body, presence of a lobed U-shaped oral sphincter, absence of a pre-pharynx, short caeca terminating at the end of the second third of body, a sinuous/coiled seminal vesicle, a pre-testicular ovary, non-intercaecal vitellarium, and a smaller egg, and it parasitizes a sparid fish (see Abid-Kachour et al. Reference Abid-Kachour, Mouffok and Boutiba2013). Species of the Megaperinae have a spinous tegument at the anterior extremity, an oral sucker lacking a partial U-shaped oral sphincter, a short pre-pharynx, long caeca that open as separate ani at the posterior extremity, a saccular seminal vesicle, a post-testicular ovary, extra- and intercaecal vitellarium, a larger egg, and they are restricted to ostraciid fishes (Blend et al. Reference Blend, Karar and Dronen2017; Bray Reference Bray, Jones, Bray and Gibson2005b; Cable Reference Cable1954; Manter Reference Manter1933, Reference Manter1934, Reference Manter1947; Nahhas and Cable Reference Nahhas and Cable1964; Pulis et al. Reference Pulis, Curran, Andres and Overstreet2014). Additionally, members of Megapera Manter, Reference Manter1934 and Thysanopharynx Manter, Reference Manter1933 possess symmetrical testes in the forebody, a post-testicular ovary, and vitellarium restricted mainly to the hindbody (Bray Reference Bray, Jones, Bray and Gibson2005b; Cable Reference Cable1954; Manter Reference Manter1933, Reference Manter1934, Reference Manter1947; Nahhas and Cable Reference Nahhas and Cable1964), whereas S. mediterraneae has median tandem testes in the mid hindbody, a pre-testicular ovary, and vitellarium extending well into the forebody (Abid-Kachour et al. Reference Abid-Kachour, Mouffok and Boutiba2013). Megapera spp. are consistent with S. mediterraneae in possessing an oral sucker much larger than the ventral sucker; however, the former are differentiated by the presence of radiating alternating muscular and cellular bands in the oral sucker. Diagnostic features of Haintestinum Pulis, Curran, Andres & Overstreet, 2014 are the most morphologically converged with S. mediterraneae—in particular, having median tandem testes with irregular margins, a pre-testicular/post-acetabular ovary in the anterior hindbody, an elongate pre-ovarian seminal vesicle, a median genital pore between the intestinal bifurcation and ventral sucker, and long vitellarium distributed from near the posterior extremity to the posterior level of the oral sucker. Species of Haintestinum differ from S. mediterraneae in having an infundibuliform-shaped oral sucker almost wholly enclosed in a fold of the body wall, H-shaped caeca, a hermaphroditic duct, and a noticeably submedian ovary as well as an oesophagus (Abid-Kachour et al. Reference Abid-Kachour, Mouffok and Boutiba2013; Pulis et al. Reference Pulis, Curran, Andres and Overstreet2014).
Sphincteristomum mediterraneae is compatible in part with members of the Apocreadiinae Skrjabin, 1942 in general body plan and position of gonads, particularly, with diagnostic features of Crassicutis Manter, Reference Manter1936 and Callohelmis Cribb & Bray, Reference Cribb and Bray1999 (see Blend et al. Reference Blend, Karar and Dronen2017; Cribb Reference Cribb, Jones, Bray and Gibson2005; Cribb and Bray Reference Cribb and Bray1999; Manter Reference Manter1936). Crassicutis spp. share with S. mediterraneae a body length less than 3 times body width, unarmed tegument, an unspecialized ventral sucker, an intestinal bifurcation in the forebody, caeca that terminate in the post-testicular region and do not reach the posterior extremity, tandem lobed testes, a pharynx smaller than ventral sucker, vitelline follicles that extend well into forebody, an I-shaped excretory vesicle extending anteriorly as far as the posterior testis, and records from the Atlantic Ocean (see Cribb Reference Cribb, Jones, Bray and Gibson2005; Manter Reference Manter1936; Pérez-Ponce de León et al. Reference Perez-Ponce de León, Razo-Mendivil, Rosas-Valdez, Mendoza-Garfias and Mejía-Madrid2008; Sparks and Thatcher Reference Sparks and Thatcher1960). However, S. mediterraneae can be differentiated from Crassicutis spp. by a highly muscular oral sucker with an inner lobed, U-shaped oral sphincter, an oral sucker more than 2 times larger than ventral one, a papillate pharynx, the absence of a pre-pharynx and oesophagus, caeca that terminate in ani, primarily extracaecal vitellarium, a sinuous/coiled seminal vesicle, a genital pore anterior to the ventral sucker by some distance, a considerably smaller egg size, a ventro- vs dorso-subterminal excretory pore and its distinct host group of sparid fishes. Concerning Callohelmis, its species exhibit several common features with S. mediterraneae—in particular, an unarmed tegument, an unspecialized ventral sucker, an intestinal bifurcation in the forebody, caeca that terminate blindly within the post-testicular region, tandem testes, a pharynx smaller than suckers, a short I-shaped excretory vesicle limited to the post-testicular region and a ventrally subterminal excretory pore (see Cribb Reference Cribb, Jones, Bray and Gibson2005; Cribb and Bray Reference Cribb and Bray1999). Sphincteristomum mediterraneae differs from Callohelmis by the same previously mentioned differential features listed for Crassicutis spp., except for the ventro-subterminal excretory pore, restriction of vitelline follicles to the hindbody and the distinct distribution of S. mediterraneae in the Atlantic and not the Pacific Ocean.
The subfamilial placement of S. mediterraneae is problematic. No apocreadiines possess a wavy/scallop-edged pharynx, a partial muscular U-/V-shaped sphincter half encircling the oral aperture postero-ventrally, or an oral sucker size ≥ 2 times the ventral sucker size. Sphincteristomum mediterraneae represents an intermediate taxonomic status that combines some distinctive features of the Megaperinae and the Schistorchiinae. Sphincteristomum mediterraneae shares with schistorchiines an oral sucker with a partial U-shaped sphincter half encircling the oral aperture postero-ventrally. However, the restricted distribution of schistorchiines to the Indo-Pacific Ocean rather than to the Atlantic Ocean, as seen for megaperines (see Blend et al. Reference Blend, Karar and Dronen2017; Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023; Mansour et al. Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025; Pulis et al. Reference Pulis, Curran, Andres and Overstreet2014; Present phylogenetic results), combined with a wavy/scallop-edged (cf. papillate) pharynx and a notably short genital atrium, suggests S. mediterraneae is morphologically closer to megaperines than schistorchiines.
Among genera of the Megaperidae, S. mediterraneae possesses a unique combination of generic-level differential features: a highly muscular oral sucker with a distinct partial U-shaped sphincter, two caeca that terminate in separate ani within the post-testicular space, two post-ovarian testes in mid hindbody, an extracaecal vitellarium extending well into forebody, a median entire ovary, and a preacetabular median genital atrium in addition to a distinct host group (sparid fishes) and considerably distant/distinct locality (East Atlantic Ocean [Mediterranean Sea]). However, the distorted and poor condition of the specimens’ illustrations, coupled with the disputed taxonomic status at the subfamily level and the inability to re-examine the original type-specimens, weakens the proposal to erect a new taxon. Given these ambiguities in concert with the absence of supportive molecular data, we provisionally assign S. mediterraneae a ‘species inquirendum’ until further studies can be performed with thorough morphological and sequence data.
On distributions of schistorchiines in the Indo-West Pacific Ocean
Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) referred to (i) the potential of even broader distributions of schistorchiine species as collection efforts become more exhaustive, and (ii) the plausible fit of the distribution of schistorchiine species within a paradigm of broad distributions of trematodes in the Indo-West Pacific Ocean within tropical marine fishes. In lacking sufficient substantial distributional evidence based on molecular data, Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) indicated that more sampling across different localities within the Indo-West Pacific Ocean was required. Our investigations show that molecular data of S. carneus (sensu lato) is compatible with its widespread distribution over a wide geographic range from Australia (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023) to the Red Sea (Mansour et al. Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025). On a narrower scale, L. aniferum (sensu lato) is distributed from off Lizard Island to Ningaloo Reef (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023).
Evidence from host-parasite data indicates the occurrence of such wide distributions of some schistorchiine species. Records of Paraschistorchis seychellesiensis are from off Seychelles (Toman Reference Toman1989) and off Heron and Lizard Islands (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023). Records of Paraschistorchis stenosoma are from off Hawaii (Hanson Reference Hanson1953; Pritchard Reference Pritchard1963; Yamaguti Reference Yamaguti1970) and the Great Barrier Reef (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023). Paraschistorchis zancli is recorded off Hawaii (Hanson Reference Hanson1953; Yamaguti Reference Yamaguti1970), French Polynesia (Lo et al. Reference Lo, Morgan, Galzin and Cribb2001), Mariana Islands (Toman and Kamegai Reference Toman and Kamegai1974), and the Society Islands (Bray et al. Reference Bray, Webster, Bartoli and Littlewood2005; Olson et al. Reference Olson, Cribb, Tkach and Littlewood2003), in addition to the Great Barrier Reef, New Caledonia, and Palau (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023). Plesioschistorchis callyodontis is reported from off South Kuroshio, Japan (Yamaguti Reference Yamaguti1942) and the Red Sea (Blend et al. Reference Blend, Karar and Dronen2017; Nagaty and Abdel Aal Reference Nagaty and Abdel Aal1962). Plesioschistorchis sigani (Yamaguti, Reference Yamaguti1942) Blend, Karar & Dronen, Reference Blend, Karar and Dronen2017 is recorded from off South Kuroshio (Yamaguti Reference Yamaguti1942) and the Celebes Sea (Yamaguti Reference Yamaguti1953). Schistorchis skrjabini is reported from the South China Sea (Parukhin Reference Parukhin and Skjrabin1963), off India in the Bay of Bengal (Hafeezullah Reference Hafeezullah1981), Moreton Bay (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023), and the Gulf of Tonkin (Parukhin Reference Parukhin1989; Truong et al. Reference Truong, Ngo, Bui, Palm and Bray2022). Sphincteristomum acollum is reported from the Gulf of Tonkin (Oshmarin et al. Reference Oshmarin, Mamaev and Parukhin1961; Parukhin Reference Parukhin1989; Truong et al. Reference Truong, Ngo, Bui, Palm and Bray2022), off Lizard Island (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023), and the South China Sea (Parukhin and Chikunova Reference Parukhin and Chikunova1964). Accordingly, we can expect the existence of a wide distribution in other schistorchiine species with more extensive sampling.
Although coupling between molecular results and host-parasite data may present ample evidence for widespread species of the Schistorchiinae, some notable exceptions challenge this generalization. First, the hypothesis of species complexes and the potential propagation of cryptic species in schistorchiines as reported in the population composition of L. aniferum (sensu lato) and S. carneus (sensu lato). Such results suggest a higher unrecognized parasite richness in the Schistorchiinae; hence, it is likely that morphology-based records of a particular schistorchiine species may include morphologically indistinguishable cryptic taxa, not of a single species, which in turn, requires a more nuanced and accurate examination. Second, Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) shed light on the considerable importance of host specificity in differentiating several schistorchiine species. Mansour et al. (Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025) pointed out that schistorchiine species exhibit a pronounced preference for host specificity over species richness, ranging from oioxenous to stenoxenous, indicating the pivotal role of host recognition in the diversification of schistorchiines, even in cases where morphological differentiation is challenging. The present study is consistent with Mansour et al. (Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025) and demonstrates the reasonable occurrence of cryptic species in some multi-record schistorchiine species. Consequently, to avoid not recognizing the potential existence of species complexes, meticulous attention must be paid when recording a species from different hosts and determining those that are morphologically and ecologically proximate compared to those that are more distant. Third, the reports supported by molecular data on the co-occurrence of two schistorchiine species within a single host referred to their close morphology with only a narrow range of differences; Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) recorded Blendiella tridecimtestis Magro, Cutmore, Carrasson & Cribb, Reference Magro, Cutmore, Carrasson and Cribb2023 and Blendiella trigintatestis Magro, Cutmore, Carrasson & Cribb, Reference Magro, Cutmore, Carrasson and Cribb2023 from the same hosts (B. undulatus and S. chrysopterum) within the same locality off Heron and Lizard Islands near Australia. Host-parasite data of schistorchiines referred to the existence of such additional previous patterns as follows: (i) Yamaguti (Reference Yamaguti1970) recorded P. stenosoma and P. zancli from the sandwich isle file, Cantherhines sandwichiensis (Quoy & Gaimard) (Tetraodontiformes: Monacanthidae), from off Hawaii; (ii) Blend et al. (Reference Blend, Karar and Dronen2017) collected P. callyodontis and P. haridis from the common parrotfish, Scarus psittacus Forsskål (Perciformes: Scaridae), from nearby localities in the northern Red Sea; (iii) Mansour et al. (Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025) phylogenetically delineated Australian records of P. stenosoma into two entities from a single host (C. pardalis) at nearby localities (Lizard Island vs Heron Island); and (iv) the present study, through morphometrical comparisons within a multivariate cluster analysis on records of L. aniferum (sensu lato) coupled with molecular data, is consistent with the presence of a species complex and differentiated these records into two distinctive groups. Based on morphological analyses of the aforementioned examples, we find that each pair of aforementioned species is close morphologically with only a narrow range of differences, which might result in an overlap in the individual combinations of species-specific diagnostic characters (particularly in the case of small-sized specimens) – hence, a doubtful/spurious record.
Testes arrangement as a characteristic in schistorchiines with multiple testes
Through a comprehensive review of the nature and arrangement of schistorchiines with multiple testes (i.e., testes no. ≥ 5), we found examples of testes that were post-ovarian, smooth to slightly indented, subglobular to ellipsoidal to transversely elongate, separated to contiguous or nearly so, and arranged either in a median column of one testis wide, in two distinct submedian elongate columns – each of one testis wide –or in a cluster initially of 2−4 testes wide but narrowing progressively and eventually to 1−2 testes wide posteriorly.
Schistorchiines with one median testicular column show variation either as a straight tandem row mainly or a zig-zag pattern occasionally. For example, Pa. longivesiculurus (Hafeezullah Reference Hafeezullah1981, Figure 1), Pa. zancli (Hanson Reference Hanson1953, Figure 2a), Pa. seychellesiensis (Toman Reference Toman1989, Figure 1a), Pa. stenosoma (Hanson Reference Hanson1953, Figure 2a; Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023, Figure 7A), Pl. haridis (Blend et al. Reference Blend, Karar and Dronen2017), and Plesioschistorchis manteri (Gupta & Tandon, Reference Gupta and Tandon1984) Blend, Karar & Dronen, Reference Blend, Karar and Dronen2017 (Gupta and Tandon Reference Gupta and Tandon1984, Figure 1) showed a straight tandem row of testes, while Pl. haridis (Blend et al. Reference Blend, Karar and Dronen2017; Nagaty Reference Nagaty1957, Figure 1) exhibited a zig-zag pattern. Within schistorchiines possessing two distinct columns of testes, the dextro-submedian column consists of a larger number of them. Such arrangement of testes is only observed in S. carneus (Blend et al. Reference Blend, Karar and Dronen2017, Figure 15; Hafeezullah Reference Hafeezullah1981, Figure 6; Johnston Reference Johnston1913, Figures 11 and 38; Lühe Reference Lühe1906, Figures 11 and 12; Madhavi et al. Reference Madhavi, Narasimhulu and Shameem1986, Figure 6; Mansour et al. Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025, Figures 1 and 2) and Schistorchis tetrodontis (Nagaty, Reference Nagaty1956) Blend, Karar & Dronen, Reference Blend, Karar and Dronen2017 (Nagaty Reference Nagaty1956, Figure 2). Regarding the clustered configuration of testes in schistorchiines, these are distributed medially/submedially into 2−3 contiguous columns, and generally the dextro-submedian one comprises the largest number of testes. For example, B. trigintatestis (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023, Figure 6A), B. tridecimtestis (Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023, Figure 6A), Neomegacreadium okinawanum Machida & Kuramochi, Reference Machida and Kuramochi1999 (Machida and Kuramochi Reference Machida and Kuramochi1999, Figure 16), Pl. callyodontis (Blend et al. Reference Blend, Karar and Dronen2017, Figure 1; Yamaguti Reference Yamaguti1942, Figure 15), Pl. sigani (Yamaguti Reference Yamaguti1942, Figure 16), and S. skrjabini (Hafeezullah Reference Hafeezullah1981, Figure 5) possess two submedially elongated contiguous columns. Only Schistorchis paruchini Kurochkin, Reference Kurochkin1974 (Kurochkin Reference Kurochkin1974, p. 67) and S. skrjabini (Hafeezullah Reference Hafeezullah1981, Figure 4; Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023, Figure 4A; Parukhin Reference Parukhin and Skjrabin1963, Figure 1) show a pattern of three contiguous and somewhat overlapping columns. In our review of the literature and voucher specimens, we did not find any evidence of intraspecific variation/fluctuation within the same species in regard to testicular arrangement, suggesting that this is a reliable differentiating character. Additionally, the differences in such testes arrangements within a population suggest the presence of two different entities at least.
Toman (Reference Toman1989) described P. seychellesiensis from the intestine of C. pardalis off the Seychelles Islands in the Indian Ocean. Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) collected schistorchiine specimens from off Heron and Lizard Islands, Australia, identifying them as P. seychellesiensis based on morphology consistent with the original description and conspecific host. Specimens of Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) exhibited a clustered configuration of testes with a longer post-testicular region compared to a median testicular column with a shorter post-testicular region in the original description, and while such a difference was controversial, Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) tentatively assigned their specimens to P. seychellesiensis, waiting for further sampling and molecular evidence from the type-location to improve confidence in this identification. Our review of the original description of P. seychellesiensis combined with a photograph examination of the type-specimen reveals several morphological differences compared to the specimens of Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023). The more significant is that P. seychellesiensis exhibits a testes arrangement in a single median column that is transversally elongated vs roughly ovoid testes, in a cluster initially 2 testes wide but narrowing progressively to 1 posteriorly, as well as a smaller post-testicular region occupying one-sixth of body length compared to a larger post-testicular region of about one-third of body length. The anterior extension of the vitellarium in P. seychellesiensis ranges from about the level of the posterior margin of the ventral sucker or slightly posterior to it without confluence of vitelline follicles in the post-testicular region compared to an anterior extent of the vitellarium in specimens of Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) from about the level of the anterior margin of the ventral sucker and confluence of vitelline follicles in the post-testicular region. Despite being reported from the same host species, C. pardalis, within the Indo-Pacific Ocean, the distant locality and distinctive ecology of the GBR compared to that of the Seychelles Islands should be well considered in light of the plausibility of the existence of associated/closely related digeneans within the same host species as well as evidence mounting regarding the presence of species complexes within some schistorchiine taxa (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023; Mansour et al. Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025; Present study). The combination of aforementioned features suggests the Australian population (P. cf. seychellesiensis) as a free-standing taxon distinct from the original description of P. seychellesiensis by Toman (Reference Toman1989); however, further reinvestigation is needed for an appropriate future description and designating type specimens (Holotype specimen at least).
On glandular components in schistorchiines possessing a muscular oral sucker
Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) suggested the probable existence of glandular development to some degree in nearly all schistorchiines based on i) presence of glandular elements in the muscular oral suckers of two species of Paraschistorchis (P. stenosoma and P. seychellesiensis) and ii) the direct association between muscular strength of the oral sphincter and developmental degree of glandular components in oral suckers of a muscular nature.
Photographs and vouchers of two species of Plesioschistorchis (P. callyodontis and P. haridis) showed minor glandular components in the oral sucker near the anterior rim, which become clearer with increasing musculature of the oral sphincter. This has been recognized in S. mediterraneae where the outer part of the oral sucker is composed of circular and longitudinal muscles as well as numerous gland-like cells (see Abid-Kachour et al. Reference Abid-Kachour, Mouffok and Boutiba2013, Figures 2a,b, 4c). Accordingly, we support the aforementioned hypothesis of Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023), who suggested a function linking the oral sphincter and glandular components of the oral sucker; the oral sphincter captures host tissue within the cavity of the oral sucker wherein secretions of glandular components initiate its digestion. In addition, we concur with Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) that the presence of glandular components within the muscular oral sucker of some schistorchiines is not as pronounced as those of schistorchiines possessing an oral sucker of a ‘complex’ nature; thus, this observation does not conflict with adopting oral sucker nature (‘simple’ vs ‘complex’) in the distinction of genera.
On relations between the Schistorchiinae and the Megaperinae
Before investigations of Pulis et al. (Reference Pulis, Curran, Andres and Overstreet2014) involving the nature and taxonomic status of megaperines, the concept of either the Megaperinae (see Manter Reference Manter1963) or the Megaperidae (see Bray Reference Bray, Jones, Bray and Gibson2005b; Manter Reference Manter1934; Yamaguti Reference Yamaguti1971) was originally restricted to diagnosing Megapera and Thysanopharynx. This earlier concept could be easily differentiated from the recent diagnosis of the Schistorchiinae (see Blend et al. Reference Blend, Karar and Dronen2017; Cribb Reference Cribb, Jones, Bray and Gibson2005) by the following features: i) an oral sucker lacking a partial U-shaped oral sphincter vs an oral sucker with a partial U-shaped sphincter half encircling the oral aperture postero-ventrally; ii) a cup-shaped pharynx with lobed anterior margin vs a non-papillate one; iii) two lateral, pre-ovarian and large testes in the forebody vs two to multiple, post-ovarian, tandem or opposite testes in the hindbody; iv) a short muscular genital atrium vs no atrium; v) a lobed post-testicular ovary vs an entire pre-testicular one; vi) opening of the distal portions of the male and female ducts into a genital atrium separately vs their merging to form a hermaphroditic duct; and vii) megaperines are distributed within the Northwestern Atlantic Ocean vs schistorchiines that are restricted to the Indo-Pacific Ocean. Schistorchiines also showed variation in tegument (spinous/unarmed), oral sucker shape (oval/globular or infundibuliform), oral sucker compared to ventral sucker size (larger/sub-equally sized/smaller), circumoral lobes (present/absent), oesophagus (short/non-evident), pre-pharynx (very short/lacking), ovary relative to testes size (larger/sub-equally sized/smaller), vitellarium distribution (restricted to hindbody/enter well into forebody); egg number (few/many); and host group (Tetraodontiformes/Perciformes). In contrast, members of Megapera and Thysanopharynx exhibit more consistency with a partially spinous tegument, an oval/globular oral sucker larger than the ventral sucker, absence of circumoral lobes or oesophagus, presence of a pre-pharynx, a lateral vitellarium restricted mainly to the hindbody or region of ventral sucker, few eggs, and a tetraodontiform host. Additionally, the ventral sucker is either equatorial or pre-equatorial and the oral sucker almost wholly enclosed in a fold of the body wall or not in Megapera and Thysanopharynx spp., whereas a pre-equatorial ventral sucker only and no fold in the body wall is seen in schistorchiines.
After establishing Haintestinum within the Megaperinae using molecular data, Pulis et al. (Reference Pulis, Curran, Andres and Overstreet2014) amended the morpho-diagnostic features of the Megaperinae to include variation in oral sucker shape from globular to funnel-shaped; presence or absence of an oesophagus; caeca with or without anterior extensions to the oral sucker; two testes positioned in either forebody or hindbody, pre-ovarian or post-ovarian, and tandem-to-oblique or opposite testes; a lobed or entire, pre- or post-testicular ovary; differences in ovary size compared to testes ranging from larger, equally sized to smaller; an anterior extent of the vitellarium reaching well into the forebody; and few to many eggs. We can add the change of the nature of the distal portions of the male and female ducts from opening into a muscular genital atrium separately to their merging into a hermaphroditic duct terminating in a shallow genital atrium. These amendments expanded the concept of the Megaperinae to be more generalized in several features when compared to their counterparts in the Schistorchiinae. Hence, the differential features of megaperines have been restricted more recently to a pharynx with a lobed anterior rim, absence of a U-shaped sphincter half encircling the oral aperture postero-ventrally, and limited distribution to the Northwest Atlantic Ocean.
Some megaperines are predicted to possess a pattern of glandular development convergent to that in some schistorchiines possessing an oral sucker of ‘simple’ type. Megapera spp. show a distinctly large oral sucker with conspicuous radiating muscle bands interspersed by bands of cells having apparently glandular components (see Bray Reference Bray, Jones, Bray and Gibson2005b, Figure 54.1; Manter Reference Manter1933, Figures 1, 3–5; Nahhas and Cable Reference Nahhas and Cable1964, Figure 15). Further, the adapted diagnosis of Paraschistorchis suggested the U-shaped partial sphincter at the aperture is either prominent or almost undetectable, yet not completely absent (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023). We noted this in some voucher specimens of Pl. haridis. Thus, we predict this general pattern in other schistorchiines having oral suckers of a ‘simple’ nature. In contrast, those having oral suckers of a ‘complex’ nature possess a strong prominent oral sphincter; its size is correlated with the increase in glandular components of the oral sucker. Further, Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) referred to the variation of oral sucker shape (rounded to elliptical vs funnel-shaped) from within the same species (e.g., Pa. zancli) and suggested this variation was not of diagnostic value in differentiating between Pa. zancli and Pa. stenosoma. This differs from Blend et al. (Reference Blend, Karar and Dronen2017) and should not be adopted as a main differential feature between these two species. As discussed earlier, schistorchiines having an oral sucker of a ‘simple’ nature exhibit variation in the clarity of the oral sphincter and in the shape of the oral sucker itself within and across species. This would remove some differential barriers between schistorchiines and megaperines, in particular, the presence, non-detectability or absence of an oral sphincter, at least superficially, highlighting convergence/overlap between the two subfamilies and requiring more careful/accurate examination in the future to define the true nature of these controversial features.
The intermediate taxonomic status of S. mediterraneae between the Megaperinae and the Schistorchiinae causes confusion and overlap among differential features and greatly reduces the distinguishing features between the two subfamilies. Considering S. mediterraneae as a member within the Schistorchiinae would (i) expand the distribution of schistorchiines to include the Eastern Atlantic Ocean (Mediterranean Sea) along with the Indo-Pacific Ocean and overlap with the limited distribution of megaperines within the Northwest Atlantic Ocean; (ii) undermine the importance of a papillate pharynx as a distinctive character within megaperines; (iii) add another diagnostic feature to schistorchiines – the short genital atrium; and (iv) amend the nature of the distal portions of the male and female ducts, either being separated or merged into a hermaphroditic duct. The only remaining characteristic for differentiating schistorchiines from megaperines would be the absence of a partial muscular U-/V-shaped sphincter half encircling the oral aperture postero-ventrally. Placement of S. mediterraneae within the Megaperinae would (i) undermine the importance of the partial muscular U-/V-shaped sphincter encircling the anterior aperture of the oral sucker as a distinctive character among schistorchiines; (ii) expand diagnosis of megaperines to include taxa having an oral sucker with or without an oral sphincter; and (iii) widen the host range of megaperines to include sparids along with ostraciids. We feel that placing S. mediterraneae within the Megaperinae will maintain a greater number of variations between the two subfamilies—specifically, a restricted distribution to the Atlantic Ocean, a papillate pharynx, and a notable short genital atrium in megaperines vs an Indo-Pacific distribution, a non-papillate pharynx, and the absence of a genital atrium in schistorchiines. Furthermore, this will ensure sufficient taxonomic utility and facilitate/streamline research and management efforts of such organisms.
Molecular phylogenetic analyses demonstrated that megaperines are more closely allied with taxa of the Schistorchiinae than with the Apocreadiinae (see Magro et al. Reference Magro, Cutmore, Carrasson and Cribb2023, Figure 3; Mansour et al. Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025, Figure 4; Pulis et al. Reference Pulis, Curran, Andres and Overstreet2014, Figure 10; Present study). Such close relationship was attributed to some shared characteristics—in particular, the possession of an anus/ani (see Mansour et al. Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025; Pulis et al. Reference Pulis, Curran, Andres and Overstreet2014) and their common association with marine tetraodontiform hosts (see Mansour et al. Reference Mansour, Karar, Hassan, Mohamadain and Abdel-Hakeem2025) vs blind-ending caeca/cyclocoel and non-tetraodontiform, marine and freshwater fishes in apocreadiines. We find the aforementioned interpretation to be partially correct for specimens included in previous phylogenetic studies as well as the present investigation, as no DNA sequences were available for three of eight schistorchiine genera we recognize that do not have a common anus/separate ani and/or parasitize tetraodontiform fish: Neomegacreadium (a uroproct/tetraodontiform host), Plesioschistorchis (blind caeca/perciform host), and Sphincterostoma (a uroproct/perciform host). Additionally, S. mediterraneae possesses separate ani and was recorded from a sparid host, while Pa. longivesiculurus was recorded from a perciform host (see Hafeezullah Reference Hafeezullah1981) and both not included in the phylogenetic analyses.
In conclusion, we find schistorchiines and megaperines share several features that exhibit the same pattern of variation (e.g., gonad appearance, distribution, and position; vitellarium extension; range of oesophagus length; oral sucker shape; glandular components of oral sucker; tegument spination) and combine to lower the number of differential characteristics between the two subfamilies. This is reflected in their obvious phylogenetic convergences. Such a close relationship can be attributed to the possibility that geographic isolation (allopatry) played a role in the division of the Schistorchiinae/Megaperinae cluster and the divergence into the two currently known subfamilies. In this context, Magro et al. (Reference Magro, Cutmore, Carrasson and Cribb2023) clarified that observed distribution patterns suggest the influence of multiple processes on the distribution of schistorchiines, and this could happen on a larger scale (i.e., schistorchiine/megaperine cluster). There is also a possibility that this convergence suggests that the two subfamilies are just one larger group (i.e., not two distinct subfamilies) and that the internal separation into two sister clades is only a result of the i) difference in geographical distribution and ii) megaperines being limited to ostraciid fishes and having a papillate pharynx. Consequently, this possibility weakens the importance of the two aforementioned features as distinguishing characteristics at the subfamily level. However, the first assumption (i.e., having two distinct subfamilies) represents the most acceptable option, as it maintains and ensures taxonomic utility and facilitates following up on such parasites. The Megaperinae and the Schistorchiinae both apparently fulfill the holophyletic requirement whereby each subfamily is always partly subjective, holophyletic and useful, reflecting some significant distinctions (see Envall Reference Envall2008; Hörandl and Stuessy Reference Hörandl and Stuessy2010). These two hypotheses are speculative and require further research and molecular studies of more specimens (particularly for species/genera not sequenced yet) from these two subfamilies and over wider geographical ranges.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X25100710.
Data availability statement
All the data generated from this study are included in the paper and can be accessed from the corresponding author upon request for approval. The nuclear DNA sequences obtained in this study have been deposited in the GenBank database.
Acknowledgements
The authors thank the Department of Zoology, Faculty of Science, South Valley University, Qena, Egypt, for providing AM and YFMK with the required facilities and help in collecting fish samples. Continued appreciation is offered by CKB to Karen Stevenson (Director) and Dillon Beal and Phoebe Cundiff (Curators), Corpus Christi Museum of Science & History, as well as to Gordon College for their indefinite loan of equipment. We also thank the reviewers for their comprehensive suggestions, edits, and comments, which were helpful, quite positive, and improved our manuscript to a wide extent. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Authors’ contribution
Yasser F.M. Karar: Conceptualization, Methodology, Formal analysis, Investigation, Writing – Original Draft, Writing – Review & Editing, Visualization. Charles K. Blend: Conceptualization, Writing – Review & Editing, Visualization, Supervision. Ali Mansour: Methodology, Writing –Review & Editing, Validation, Investigation
Financial support
None.
Competing interests
The authors declare that they have no conflict of interest.
Ethical standard
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. South Valley University ethics approval no. REC-FScSVU:03/12/23.
Use of artificial intelligence (AI) tools
The authors declare that there is no use of AI-assisted technologies in text/images generation or to analyse or extract insights from data or other material.
