Syst Parasitol (2023) 100:381–413 https://doi.org/10.1007/s11230-023-10093-5 (0123456789().,-volV) ( 01234567 89().,-volV) Integrated characterisation of nine species of the Schistorchiinae (Trematoda: Apocreadiidae) from Indo-Pacific fishes: two new species, a new genus, and a resurrected but ‘cryptic’ genus Lori Magro . Scott C. Cutmore . Maite Carrasson . Thomas H. Cribb Received: 31 October 2022 / Accepted: 12 April 2023 / Published online: 10 May 2023  The Author(s) 2023 Abstract We report nine species of the Schistorchi- aniferum Manter, 1963, previously a synonym of Sph. inae Yamaguti, 1942 (Apocreadiidae Skrjabin, 1942) acollum. Lobatotrema aniferum is phylogenetically from Indo-Pacific marine fishes. Molecular data (ITS2 distant from, but morphologically similar to, Sph. and 28S rDNA and cox1 mtDNA) are provided for all acollum and Lobatotrema is recognised as a ‘cryptic species and the genus-level classification of the genus’. We propose Blendiella n. gen. for B. trigin- subfamily is revised. For Schistorchis Lühe, 1906, tatestis n. sp. and B. tridecimtestis n. sp. These species we report the type-species Sch. carneus Lühe, 1906 are broadly consistent with the present morphological and Sch. skrjabini Parukhin, 1963. For Sphinteristo- concept of Schistorchis but are phylogenetically mum Oshmarin, Mamaev & Parukhin, 1961 we report distant from the type-species; a larger number of the type-species, Sph. acollum Oshmarin, Mamaev & testes and some other subtle morphological characters Parukhin, 1961. We report and re-recognise Loba- in species of Blendiella serve to distinguish the two totrema Manter, 1963, for the type and only species, L. genera. We report three species of Paraschistorchis Blend, Karar & Dronen, 2017: P. stenosoma (Hanson, 1953) Blend, Karar & Dronen, 2017 (type-species), P. This article is registered in the Official Register of Zoological seychellesiensis (Toman, 1989) Blend, Karar & Dro- Nomenclature (ZooBank) as urn: nen, 2017, and P. zancli (Hanson, 1953) Blend, Karar lsid:zoobank.org:pub:0C8BBC3C-0EC4-4C48-BFA8- 9EDCB129B58A. This article was published as an Online First & Dronen, 2017. Lobatotrema aniferum, P. steno- article on the online publication date shown on this page. The soma, and Sch. carneus each have two distinct cox1 article should be cited by using the doi number (10.1007/ populations either over geographical range or in s11230-023-10093-5). This is the Version of Record. sympatry. Available evidence suggests that most of these species, but not all, are widespread in the tropical L. Magro  T. H. Cribb (&) School of Biological Sciences, The University of Indo-Pacific. Queensland, St Lucia, QLD 4072, Australia e-mail: t.cribb@uq.edu.au Introduction S. C. Cutmore Queensland Museum, Biodiversity and Geosciences Program, South Brisbane, QLD 4101, Australia Members of the Apocreadiidae Skrjabin, 1942 para- sitise a wide range of marine and freshwater fishes, M. Carrasson with the family comprising four subfamilies (the Departament de Biologia Animal, de Biologia Vegetal i d’Ecologia, Universitat Autònoma de Barcelona, Apocreadiinae Skrjabin, 1942; Megaperinae Manter, Cerdanyola del Vallès, 08193 Barcelona, Spain 1934; Postporinae Yamaguti, 1958; and 123 382 Syst Parasitol (2023) 100:381–413 Schistorchiinae Yamaguti, 1942) and 23 genera individual worms preserved in 80% ethanol were (Blend et al., 2017). The family name is presently processed for parallel morphological and molecular controversial. Blend et al. (2017) proposed that the analyses (hologenophores and paragenophores sensu senior synonym is Megaperidae Manter, 1934, but Pleijel et al., 2008). commentary in WoRMS (2022) argues for the reten- tion of Apocreadiidae (citing that the change was non- Morphology compliant with Article ICZN Article 35.5). For the present, we accept the latter, arguably conservative Trematodes for morphological examination were view. Morphological characters uniting species within washed in distilled water, stained in Mayer’s haema- this family include an I-shaped excretory vesicle, lack toxylin, destained in 1.0% hydrochloric acid, and of a cirrus-sac, extensive vitelline follicles, dispersed neutralised in 1.0% ammonium hydroxide. Worms eye-spot pigment in the forebody, and the genital pore were then dehydrated in a graded ethanol series, opening immediately anterior or (rarely) posterior to cleared in methyl salicylate, and mounted on slides in the ventral sucker (Cribb & Bray, 1999). Although this Canada balsam. Measurements were taken from a live morphology is far from distinctive, molecular phylo- feed produced with an Olympus SC50 digital camera genetic analysis has consistently shown the family to attached to an Olympus BX-53 compound microscope be monophyletic and indeed worthy of recognition as with cellSens v1.13 software. Drawings were made the sole family in the suborder Apocreadiata (Olson using a drawing tube connected to the same micro- et al., 2003; Pérez-Ponce de León & Hernandez-Mena, scope and subsequently digitised using a drawing pad 2019). and Adobe Illustrator CC 2018. Figures are presented The subfamily Schistorchiinae is presently recog- for species or combinations not previously reported nised within the Apocreadiidae by the possession of an from Australia. All measurements are in micrometres unusual and distinctive U-shaped partial sphincter and given as the range, followed by the mean in embedded within the oral sucker (Cribb, 2005; Pulis parentheses. The following abbreviations are used: et al., 2014). Following recent revision by Blend et al. MNHN, Museum National d’Histoires Naturelles, (2017), the subfamily is recognised as comprising six Paris, France; QM, Queensland Museum, Brisbane, genera and 17 species, all of which infect fishes of the Australia; WAM, Western Australian Museum, Perth, Indo-west Pacific except for the enigmatic species Australia. To comply with the recommendations set Sphincteristomum mediterraneae Abid-Kachour, out in article 8.5 of the amended 2012 version of the Mouffok & Boutiba, 2013, which parasitises sparid International Code of Zoological Nomenclature fishes in the Mediterranean Sea (Abid-Kachour et al., (ICZN, 2012), details of the new species have been 2013). submitted to ZooBank and registered with Life Here we report on schistorchiines from Indo-Pacific Science Identifiers (LSID), which are provided in the fishes incorporating multi-loci molecular data that taxonomic summaries. enables independent consideration of the genus-level classification proposed by Blend et al. (2017). Molecular analyses Specimens for molecular analysis were processed Materials and methods according to the protocols used by Cribb et al. (2022). Three genetic markers were sequenced, the second Collecting internal transcribed spacer region (ITS2 rDNA), the large (28S) ribosomal subunit RNA coding region and Fishes were collected by spear, seine net, tunnel net or the cytochrome c oxidase subunit 1 (cox1 mtDNA) line from localities off Australia, French Polynesia, mitochondrial region. The complete ITS2 region was New Caledonia, and Palau. Digeneans were collected amplified and sequenced using the primers 3S (Mor- from freshly killed fish as described by Cribb & Bray gan & Blair, 1995) and ITS2.2 (Cribb et al., 1998), the (2010), fixed by being pipetted into nearly boiling partial D1-D3 28S region using LSU5 (Littlewood, saline, and immediately preserved in either formalin 1994), 300F (Littlewood et al., 2000), ECD2 (Little- (early work) or 80% ethanol (recent work). Some wood et al., 1997) and 1500R (Snyder & Tkach, 2001) 123 Syst Parasitol (2023) 100:381–413 383 and the partial cox1 region using Dig_cox1Fa (Wee support was estimated by performing 10,000 bootstrap et al., 2017) and Dig_cox1R (Wee et al., 2017). replicates. Geneious version 10.2.6 (Kearse et al., 2012) was The partial 28S sequences generated during this used to assemble and edit contiguous sequences, and study were aligned with sequences of related apocre- the start and end of the ITS2 region were determined adiids from GenBank (Table 1) using MUSCLE by annotation through the ITS2 Database using the version 3.7 (Edgar, 2004) run on the CIPRES portal ‘Metazoa’ model (Keller et al., 2009; Ankenbrand (Miller et al., 2010), with ClustalW sequence weight- et al., 2015). ing and UPGMB clustering for iterations 1 and 2. The ITS2 and cox1 sequence data generated during this resultant alignment was refined using MESQUITE study were each aligned with MUSCLE in MEGA 7 (Maddison & Maddison, 2021); the ends of the (Kumar et al., 2016) using UPGMA clustering for alignment were trimmed and ambiguously aligned iterations 1 and 2. Only two other schistorchiine ITS2 regions removed, leaving a final trimmed dataset of sequences were available on GenBank for inclusion in 1,275 bp. the analysis [AF392435–36; Lo et al. (2001)]; no Bayesian inference and maximum likelihood anal- comparable cox1 sequences were available. The ends yses of the 28S dataset were conducted to explore of each ITS2 fragment were trimmed for a final dataset relationships among these taxa. Bayesian inference of 481 base positions (bp). The cox1 alignment was analysis was performed using MrBayes version 3.2.7a transferred to Mesquite v.3.31 (Maddison & Mad- (Ronquist et al., 2012) and maximum likelihood dison, 2021), translated (echinoderm/flatworm mito- analysis using RAxML version 8.2.12 (Stamatakis, chondrial code) and inspected for internal stop codons. 2014), both run on the CIPRES portal. The best After the correct reading frame was determined, the nucleotide substitution model was estimated using first column was then removed so that the reading jModelTest version 2.1.10 (Darriba et al., 2012); both frame began on position one, simplifying position- the Akaike Information Criterion (AIC) and Bayesian coding in downstream analyses. The final cox1 dataset Information Criterion (BIC) predicted the TVM?I?C was 474 bp. All codon positions in the cox1 dataset model as the best estimator and Bayesian inference were evaluated for substitution saturation, as well as and maximum likelihood analyses were conducted non-stationarity caused by base composition bias. using the closest approximation to this model. Nodal Substitution saturation was assessed using the ‘‘Test of support in the maximum likelihood analysis was substitution saturation by Xia et al.’’ function (Xia estimated by performing 1000 bootstrap pseudorepli- et al., 2003; Xia & Lemey, 2009) as implemented in cates. Bayesian inference analysis was run over DAMBE v. 7.2 (Xia, 2018); no significant substitution 10,000,000 generations (ngen = 10,000,000) with saturation was detected. Non-stationarity was assessed two runs each containing four simultaneous Markov using the v2 function in PAUP v. 4.0 (Swofford, Chain Monte Carlo (MCMC) chains (nchains = 4) and 2002); significant non-stationarity was not detected. every 1,000th tree saved. Bayesian inference analysis Thus, all codons in the cox1 dataset were used in used the following parameters: ‘‘nst = 6’’, ‘‘rates = downstream analyses. Pairwise differences were esti- invgamma’’, ‘‘ngammacat = 4’’, and the priors mated for each dataset using the following conditions: parameters of the combined dataset were set to ‘‘ratepr ‘‘Variance Estimation Method = None’’, ‘‘Model/ = variable’’. Samples of substitution model parame- Method = No. of differences’’ and ‘‘Substitutions to ters, and tree and branch lengths were summarised Include = d: Transitions ? Transversions’’ and ‘‘Gaps/ using the parameters ‘‘sump burnin = 3,000’’ and Missing Data Treatment = Pairwise deletion’’. ‘‘sumt burnin = 3,000’’. Emprostiotrematids and Unrooted Neighbour joining analyses were conducted haploporids were designated as outgroup taxa, based using MEGA 7 for each dataset to explore species on relationships inferred in Pérez-Ponce de León & boundaries, with the following parameters: ‘‘Model/ Hernandez-Mena (2019). Method = No. of differences’’, ‘‘Substitutions to Include = d: Transitions ? Transversions’’, ‘‘Rates Species recognition among Sites = Gamma Distributed’’ and ‘‘Gaps/ Missing Data Treatment = Pairwise deletion’’. Nodal Putative species were initially identified as operational taxonomic units (OTUs) based on both morphological 123 384 Syst Parasitol (2023) 100:381–413 Table 1 Collection and accession data for Apocreadiidae species from GenBank incorporated in the 28S analyses Species Host species GenBank Reference accession # Apocreadiinae Skrjabin, 1942 Callohelmis pichelinae Cribb & Bray, 1999 Hemigymnus melapterus FJ788495 Bray et al. (2009) (Bloch) Crassicutis cichlasomae Manter, 1936 Mayaheros JQ389858 Pérez-Ponce de urophthalmus León et al. (2012) (Günther) Crassicutis manteri Pantoja, Scholz, Luque & Perez-Ponce de Satanoperca jurupari MW692063 Pantoja et al. (2021) León, 2021 (Heckel) Homalometron armatum (MacCallum, 1895) Manter, 1947 Lepomis microlophus KC710976 Curran et al. (2013a) (Günther) Homalometron avis Hernández-Mena, Cabañas-Granillo, Medina- Eugerres plumieri ON814858 Hernandez-Mena Hernández & Pérez-Ponce de León, 2022 (Cuvier) et al. (2022) Homalometron cupuloris (Ramsey, 1965) Cribb & Bray, 1999 Lepomis microlophus KT823420 Fayton et al. (2016) Homalometron elongatum Manter, 1947 Gerres cinereus HM038037 Parker et al. (2010) (Walbaum) Homalometron frocioneae Fayton & Andres in Fayton, Curran, Fundulus diaphanus KT823419 Fayton et al. (2016) Andres, Overstreet & McCallister, 2016 (Lesueur) Homalometron manteri (Overstreet, 1970) Cribb & Bray, 1999 Leiostomus xanthurus JX400852 Curran et al. Lacepède (2013b) Homalometron octopapillatum Perez-Ponce de Leon, Razo- Mayaheros beani JQ389863 Pérez-Ponce de Mendivil & Garcia-Magana, 2012 (Jordan) León et al. (2012) Homalometron pallidum Stafford, 1904 Fundulus heteroclitus HM038043 Parker et al. (2010) (Linnaeus) Homalometron palmeri Curran, Tkach & Overstreet, 2013a, 2013b Micropogonias JX400853 Curran et al. undulatus (Linnaeus) (2013b) Homalometron pseudopallidum Martorelli, 1986 Australoheros facetus JX400856 Curran et al. (Jenyns) (2013b) Homalometron robisoni Fayton & McAllister in Fayton, Curran, Fundulus notatus KT823418 Fayton et al. (2016) Andres, Overstreet & McCallister, 2016 (Rafinesque) Homalometron synagris (Yamaguti, 1953) Cribb & Bray, 1999 Scolopsis monogramma AY222243 Olson et al. (2003) (Cuvier) Neoapocreadium splendens Cribb & Bray, 1999 Scolopsis monogramma AY222242 Olson et al. (2003) Megaperinae Manter, 1934 Haintestinum amplum Pulis, Curran, Andres & Overstreet, 2014 Acanthostracion KF733447 Pulis et al. (2014) quadricornis (Linnaeus) Megapera gyrina (Linton, 1907) Manter, 1934 Acanthostracion KF733448 Pulis et al. (2014) quadricornis Megapera orbicularis (Manter, 1933) Manter, 1947 Acanthostracion KF733450 Pulis et al. (2014) quadricornis Thysanopharynx elongatus Manter, 1933 Acanthostracion KF733479 Pulis et al. (2014) quadricornis Schistorchiinae Yamaguti, 1942 Paraschistorchis zancli (Hanson, 1953) Blend, Karar & Dronen, Zanclus cornutus AY222240 Olson et al. (2003) 2017 (Linnaeus) Outgroup taxa Atractotrematidae Yamaguti, 1939 Atractotrema sigani Durio & Manter, 1969 Siganus lineatus AY222267 Olson et al. (2003) (Valenciennes) 123 Syst Parasitol (2023) 100:381–413 385 Table 1 continued Species Host species GenBank Reference accession # Isorchis cannoni Huston, Cutmore & Cribb, 2017 Siganus lineatus MF803154 Huston et al. (2018) Isorchis currani Andres, Pulis & Overstreet, 2016 Selenotoca multifasciata MF803157 Huston et al. (2018) (Richardson) Haploporidae Nicoll, 1914 Hapladena acanthuri Siddiqi & Cable, 1960 Acanthurus chirurgus MH244119 Andres et al. (2018) (Bloch) Hapladena nasonis Yamaguti, 1970 Naso unicornis AY222265 Olson et al. (2003) (Forsskål) and molecular distinctions. Morphological OTUs Oshmarin, Mamaev & Parukhin, 1961 is recognised: were assigned to genera using the classification of Sphinteristomum acollum Oshmarin, Mamaev & Blend et al. (2017). Molecular OTUs were distin- Parukhin, 1961 (the type-species). One species of guished based on nucleotide site similarity in each Lobatotrema Manter, 1963 is recognised: Loba- aligned cox1, ITS2 and 28S dataset. The final species totrema aniferum Manter, 1963 (the type- and only recognition hypothesis is proposed relative to the species); the genus and species are presently consid- criteria proposed by Bray et al. (2022), incorporating ered synonyms of Sphinteristomum and Sph. acollum. an integrated interpretation of morphology, host- A new genus, Blendiella n. gen., is proposed for two specificity, and molecular data. new species consistent with the present diagnosis for Schistorchis Lühe, 1906 but for which we recognise subtle distinctions. The two new species are easily Results distinguished as novel, based on commonly invoked schistorchiine interspecific differences, especially Overview testis number. Three known species of Paraschis- torchis Blend, Karar & Dronen, 2017 are recognised: Specimens consistent with the concept of the Schis- Paraschistorchis stenosoma (Hanson, 1953) Blend, torchiinae were collected from four families of the Karar & Dronen, 2017; P. seychellesiensis (Toman, Tetraodontiformes (Balistidae, Monacanthidae, 1989) Blend, Karar & Dronen, 2017; and P. zancli Tetraodontidae and Triacanthidae) and one of the (Hanson, 1953) Blend, Karar & Dronen, 2017. Seven Acanthuriformes (Zanclidae). Our analyses of further of the species are reported from Australian waters for samples from scarids and siganids are inconclusive the first time. and those specimens, together with those of several rare species, are reserved for future publication Molecular data dependent on further collections. Here we report on nine species, including two proposed as new. Neighbour joining analysis of the cox1 dataset distin- guished the nine morphotypes listed above at 56–117 Morphology bp (Fig. 1). Replicate sequences were produced for eight of the species (for all except Sph. acollum) and, Morphological examination distinguished nine rela- for five of these, intraspecific variation was insignif- tively clear morphotypes, initially interpreted as icant, ranging from only 0–4 bp. In contrast, three relating to three of the genera recognised by Blend morphotypes incorporated deep divisions in cox1 et al. (2017). Two known species of Schistorchis Lühe, sequence data. Sequences from specimens consistent 1906 are recognised: Schistorchis carneus Lühe, 1906 with L. aniferum from Ningaloo Reef and from off (the type-species) and Schistorchis skrjabini Parukhin, Lizard Island differed at 22 bp. Sequences from 1963. One known species of Sphinteristomum specimens consistent with P. stenosoma collected 123 386 Syst Parasitol (2023) 100:381–413 123 Syst Parasitol (2023) 100:381–413 387 b Fig. 1 Phylogram from the unrooted Neighbour-joining anal- and broadly by distinct host distributions. There are ysis of the Schistorchiinae cox1 mtDNA dataset. Bootstrap just three minor issues, all relating to cox1 variation support values are shown at the nodes, with values of \85 not shown. The scale-bar indicates the number of base differences. between morphologically indistinguishable samples. Abbreviations: G1, genotype 1; G2, genotype 2. In addition to the absence of meaningful morpholog- ical variation, for none of these combinations, with one possible exception, is there a meaningful host distinction. First, the variation in sequences from from C. pardalis from off both Heron and Lizard specimens consistent with L. aniferum from off Lizard Islands differed at 48 bp. Sequences from specimens Island and Ningaloo Reef is interpreted as intraspecific consistent with Sch. carneus from Arothron stellatus geographical variation of a kind that is now being and A. manilensis, all from off the Queensland coast, reported frequently (e.g. McNamara et al., 2014; Cribb differed at 48–49 bp. et al., 2022; Cutmore & Cribb, 2022; Wee et al., 2022). Neighbour joining analysis of the ITS2 dataset Secondly, the deep cox1 variation in sympatry seen for distinguished the nine morphotypes by 3–51 bp Great Barrier Reef (GBR) specimens consistent with (Fig. 2). The two easily distinguishable species of P. stenosoma was not replicated in ITS2 or 28S rDNA Blendiella differed at only 3 bp in the ITS2 analysis sequences. We recognise both forms as a single whereas they differed at 56–57 bp in the cox1 analysis. species; comparable sympatric and morphologically The next most similar combination of putative species indistinguishable populations have been reported for differed at 12 bp (Sph. acollum v. the two Blendiella two species of Preptetos Pritchard, 1960 on the GBR species). The six ITS2 sequences relating to P. (Bray et al., 2022). Thirdly, specimens morphologi- stenosoma were all identical, in contrast to the cox1 cally consistent with Sch. carneus from off the analysis in which they differed at 48 bp. The five Queensland coast create the greatest difficulty because sequences of L. aniferum from Ningaloo Reef differed distinctions in cox1 sequences were associated with from the six from off Lizard Island by just 1 bp in the specimens from different species of Arothron. ITS2 dataset, whereas they differed at 22 bp in the Although this combination of evidence creates a cox1 dataset. The four sequences relating to Sch. prima facie case for recognition of two species, we carneus from A. stellatus differed from the two from conclude that it is based on too few specimens to A. manilensis by 3 bp (the same as for the two species justify the recognition of cryptic species and thus take of Blendiella), whereas they differed by 48 bp in the the conservative approach of recognising a single cox1 analysis. species; the issue is considered in further detail below. Twelve newly generated 28S rDNA sequences (selected based on distinctiveness in both cox1 and Phylogenetic analysis ITS2 datasets) were used principally to explore phylogenetic relationships (see below). In terms of Bayesian inference and maximum likelihood analyses species delineation, combinations of the nine mor- of 28S rDNA dataset yielded identical topologies phospecies differed at 8–116 bp; the lowest distinction (Fig. 3), with robust support for the three apocreadiid of 8 bp was between the two new species of Blendiella. subfamilies included in the analysis and the Schis- The three morphospecies for which cox1 data demon- torchiinae as sister to the Megaperinae. Within the strated deep divisions (L. aniferum, P. stenosoma, and schistorchiine clade, the type-species of the type- Sch. carneus) all had identical 28S sequences for genus, Sch. carneus, formed a strongly-supported corresponding specimens. clade with Sch. skrjabini, but not with the two new species from Lizard Island balistids that have mor- Species recognition hypothesis phology broadly consistent with that of Schistorchis as diagnosed by Blend et al. (2017). Instead, these two Delineation of the species considered here is relatively species form a well-supported clade with Sph. acollum straightforward in the light of the species recognition from which, however, they are dramatically morpho- criteria proposed by Bray et al. (2022). The concept of logically distinct. A new genus, Blendiella, is pro- all nine morphotypes as representing distinct species is posed for them; morphological differences between supported specifically by clear molecular distinctions the Schistorchis and Blendiella species are mainly 123 388 Syst Parasitol (2023) 100:381–413 subtle. Sequences of specimens here identified as L. 6b. Caeca terminate blindly or at separate aniferum from off Lizard Island and Ningaloo Reef ani……………….………………………………….7. formed a clade sister to all those mentioned above, quite distant from Sph. acollum, a species which it 7a. Caeca end blindly……………………....Ple- resembles and with which it has previously been sioschistorchis Blend, Karar & Dronen, 2017. synonymised. Lobatotrema aniferum is thus consid- 7b. Caeca open at marginal or submarginal ani…… ered valid, rather than a synonym of Sph. acollum, and ...….Paraschistorchis Blend, Karar & Dronen, 2017. phylogenetic analysis supports the re-recognition of the genus Lobatotrema. Although these two species Apocreadiidae Skrjabin, 1942 are morphologically distinguishable, the genus Loba- Schistorchiinae Yamaguti, 1942 totrema is recognised as morphologically cryptic Schistorchis Lühe, 1906 relative to Sphincteristomum. Finally, sequences relat- Type-species: Schistorchis carneus Lühe, 1906, by ing to three species of Paraschistorchis, P. seychelle- original designation siensis, P. stenosoma and P. zancli, formed a strongly supported clade sister to all other sequenced Diagnosis schistorchiines. In view of the revised genus-level classification With characters of Schistorchiinae sensu Blend et al. mentioned above, we here propose a new key to the (2017). Body elongate to elliptical. Tegument spinous. eight genera of the Schistorchiinae. Eye-spot pigment dispersed in forebody. Pre-oral lobe present. Oral sucker highly glandular, round in Key to genera of Schistorchiinae outline; U-shaped partial sphincter at aperture promi- 1a. Testes two…………………………………..…..2. nent. Ventral sucker round in outline, smaller than oral sucker. Oesophagus absent. Intestinal bifurcation 1b. Testes [two………………..……….…..………4. immediately posterior to pharynx, anterior to ventral 2a. Caeca unite via uroproct………..…………...….... sucker. Intestinal caeca open via separate ani at ……………………Sphincterostoma Yamaguti, 1937. posterior end of body. Testes normally 11 (rarely fewer), entire, extending posteriorly from near poste- 2b. Caeca open independently via separate rior margin of ventral sucker in median cluster or in ani…………….………………………………….…3. two distinct elongate columns. Ovary entire, dextral or almost median in anterior hindbody, contiguous with 3a. Parasites of species of Balistoides and Pseudobal- anterior testes. Vitelline follicles distributed from near istes (Balistidae) ………...Lobatotrema Manter, 1963. posterior extremity to anywhere from anterior hind- 3b. Parasites of species of Abalistes and Rhinecanthus body to posterior forebody, confluent in post-testicular (Balistidae) and Sparidae…………………....Sphinc region. Excretory vesicle I-shaped, terminates close to teristomum Oshmarin, Mamaev & Parukhin, 1961. posterior margin of posterior testis. Excretory pore terminal. In intestine of monacanthid, tetraodontid and 4a. Oral sucker highly glandular……………………5. triacanthid fishes in the Indo-Pacific. 4b. Oral sucker normally muscular, or not strongly Type-species: Schistorchis carneus Lühe, 1906. glandular………………………………………..…..6. Other species: Schistorchis paruchini Kurochkin, 5a. Testes 8–11, normally in two distinct columns; 1974; Schistorchis skrjabini Parukhin, 1963; Schis- body robust……………….. Schistorchis Lühe, 1906. torchis tetraodontis (Nagaty, 1956) Blend, Karar & Dronen, 2017 5b. Testes C11, normally mainly in single column; body slender…………………...…..Blendiella n. gen. Schistorchis carneus Lühe, 1906 6a. Caeca open via uroproct.……….….… Syn. Pleorchis oligorchis Johnston, 1913 Neomegacreadium Machida & Kuramochi, 1999. 123 Syst Parasitol (2023) 100:381–413 389 123 390 Syst Parasitol (2023) 100:381–413 b Fig. 2 Phylogram from the unrooted Neighbour-joining anal- Table 2 Measurements of Schistorchis carneus. Percentages ysis of the Schistorchiinae ITS2 rDNA dataset. Bootstrap are given as a proportion of body length. support values are shown at the nodes, with values of \85 not shown. The scale-bar indicates the number of base differences. Locality Lizard Moreton Mackay cox1 genotypes for Paraschistorchis stenosoma sequences Island Bay Harbour shown where known. Abbreviations: G1, genotype 1; G2, n 2 2 2 genotype 2. Host A. stellatus A. stellatus A. manilensis Body L 5551–5558 8150–9136 7255–7579 Type-host: Arothron stellatus (Bloch & Schneider), Body W 2915–3231 3194–3473 2618–2624 Stellate puffer (Tetraodontiformes: Tetraodontidae). Body W % 52.4–58.2 38.0–39.2 34.5–36.2 Type-locality: South Modragam Paar, Sri Lanka. Forebody 1306–1477 1622–1663 1266–1449 Forebody % 23.5–26.6 18.2–19.9 17.5–19.1 New material Pre-oral L 79–81 262–348 85–90 Hosts: Arothron manilensis (Marion de Procé), Nar- OS L 857–1007 1133–1172 903–921 rowlined puffer; A. stellatus (Tetraodontiformes: OS W 1102–1131 1101–1284 878–893 Tetraodontidae). OS L % 15.4–18.1 12.4–14.4 11.9–12.7 Localities: off Lizard Island (1440’S, 14527’E), Prepharynx L 0 0 0 northern GBR; Mackay Harbour (2106’S, 14913’E), Pharynx L 137–172 284–360 262–268 north Queensland; Moreton Bay (2725’S, 15314’E), Pharynx W 461–530 537–639 429–459 south-east Queensland, Australia. VS L 519–526 637–766 504–537 Site in host: Intestine. VS W 411–478 679–710 546–606 Prevalence: off Lizard Island: 1 of 3 (33%) A. VS L / OS L 0.52–0.61 0.54–0.68 0.55–0.59 stellatus. Mackay Harbour: 1 of 1 A. manilensis. VS W / OS W 0.36–0.43 0.55–0.62 0.61–0.69 Moreton Bay: 3 of 5 (60%) A. stellatus. VS to ant. 1008–1100 499–626 1050–1072 Deposition of specimens: QM G240397–406. testis Representative DNA sequences: Partial cox1 mtDNA, Testes no. 11 11 11 seven sequences (four submitted to GenBank, Ant. testis L 384–403 372–403 309–318 OQ445523–26); ITS2 rDNA, six sequences (three Ant. testis W 380–401 258–282 308–316 submitted to GenBank, OQ442916–18); partial 28S Posttest. L 1830–2501 3401–5054 4301–4406 rDNA, three sequences (two submitted to GenBank, Posttest. L % 33.0–45.0 41.7–55.3 58.1–59.3 OQ442904–05). Ovary L 206–242 472–485 443–465 Measurements: Table 2. Ovary W 266–277 362–497 424–426 Vitelline field 3147–4131 6086–6971 5843–6076 Remarks L Previtelline L 2111–2180 1831–1981 1799–1821 This species (the type-species of the type-genus of the Postvitelline L 310–317 168–184 281–289 Schistorchiinae) was described by Lühe (1906) from Egg L 72–85 70–87 75–80 Arothron stellatus off Sri Lanka. It was subsequently Egg W 38–44 38–42 41–42 reported from Arothron hispidus (Linnaeus) by John- ston (1913) as Pleorchis oligorchis in the family Fasciolidae Railliet, 1895, from an unspecified local- New specimens reported from the type-host, from ity off north Queensland without reference to Sch. the northern GBR and Moreton Bay, and from A. carneus. Odhner (1928) recognised P. oligorchis as a manilensis from off Mackay are morphologically synonym of Sch. carneus, a proposal accepted by all consistent with previous descriptions of this species subsequent workers. The species has also been and are immediately distinct from all other described reported by Hafeezullah (1981) from A. hispidus from species of Schistorchis in their massive bodies. We the Gulf of Manaar, India and by Madhavi et al. (1986) conclude that the sequences reported here for speci- from Lagocephalus lunaris (Bloch & Schneider) mens from the type-host (but not from the type- (Tetraodontidae) from the Bay of Bengal, India. locality) serve to establish the molecular identity of 123 Syst Parasitol (2023) 100:381–413 391 the type-species of the type-genus for the Type-host: Triacanthus biaculeatus (Bloch), Short- Schistorchiinae. nosed tripodfish (Tetraodontiformes: Triacanthidae) Sequence data are available for specimens here [as Triacanthus brevirostris Temminck & Schlegel]. identified as Sch. carneus from A. stellatus from off Type-locality: South China Sea. Lizard Island and from Moreton Bay and from A. manilensis from Mackay Harbour, almost midway New material between the two other sites. cox1 sequence data for Hosts: Tripodichthys angustifrons (Hollard), Black- samples from the two fish species differ at 48 bp, ITS2 flag tripodfish (Tetraodontiformes: Triacanthidae). sequence data differ at 3 bp, and 28S sequence data are Locality: Moreton Bay (2725’S, 15314’E), south- identical. The 48 bp cox1 distinction, although sub- east Queensland, Australia. stantial, is less than that between the most similar Site in host: Intestine. combination of species recognised here (56–57 bp), the Prevalence: 13 of 57 (23%). two new species of Blendiella, and identical to the 48 Deposition of specimens: QM G240407–22. bp difference between what is below interpreted as two Representative DNA sequences: Partial cox1 sympatric populations of P. stenosoma. The ITS2 mtDNA, five sequences (three submitted to GenBank, distinction of 3 bp is identical to that between the two OQ445527–29); ITS2 rDNA, four sequences (one species of Blendiella, and greater than any distinction submitted to GenBank, OQ442919); partial 28S interpreted as intraspecific (e.g. the two cox1 popula- rDNA, four sequences (one submitted to GenBank, tions of P. stenosoma are identical). The absence of OQ442906). distinction between 28S sequences contrasts with a Measurements: Table 3. difference of 8 bp between the two species of Blendiella which are otherwise by far the most similar Description (Fig. 4A) combination of species recognised in this data set for that marker. These data present a host-associated [Based on 16 wholemount specimens, including 3 distinction in the molecular characterisation of these hologenophores.] Body robust, with maximum forms, but we have been unable to detect a consistent breadth in mid-hindbody and posterior end more morphological difference between specimens from A. sharply tapered than anterior end. Tegument spinous manilensis and A. stellatus. These data are problem- to level of ventral sucker. Pre-oral lobe strongly atic. Strictly, based on the species recognition criteria developed. Eye-spot pigment dispersed in forebody. that we employ, combined molecular and host distinc- Oral sucker round in outline, highly glandular; tion creates a basis for the recognition of separate U-shaped muscular sphincter at oral aperture promi- species. However, we choose not to propose a new nent. Prepharynx short. Pharynx unspecialised, species because of three qualitative weaknesses in the rounded, typically partly dorsal to posterior margin case. First, the molecular distinction is generally lower of oral sucker. Oesophagus absent. Intestinal bifurca- than that typically seen between combinations of tion immediately posterior to pharynx, close to level of species in this family. Secondly, the host distinction is anterior margin of ventral sucker. Caeca open at marginal in that other records interpreted as S. carneus separate ani close to posterior extremity. Ventral incorporate further host richness (Arothron hispidus sucker round in outline, unspecialised, far smaller than and Lagocephalus inermis) which either casts doubt on oral sucker. Testes 11 (10 in one specimen), entire, in the importance of host distinction or might suggest that compact group up to three testes wide and five testes even more unrecognised richness is present in the long, close to ventral sucker; post-testicular region genus. Finally, there is only a single collection of two occupying approximately two-fifths to half body specimens from a single A. manilensis, so that there is length. Seminal vesicle large, saccular, dextrally negligible replication of the basis of the key distinction antero-dorsal to ventral sucker. Genital pore ventro- in the data set. We conclude that the conservative submedian, immediately anterior to ventral sucker. option is to identify all these specimens as S. carneus Ovary entire, ovoid, median, immediately anterior to pending the gathering of further evidence. most anterior median testis, usually partly overlaps ventral sucker dorsally. Canalicular seminal recepta- Schistorchis skrjabini Parukhin, 1963 cle not detected. Vitelline follicles extend from level 123 392 Syst Parasitol (2023) 100:381–413 Table 3 Measurements of Schistorchis skrjabini and Sphinc- testes then separated into four variably distinct teristomum acollum. Percentages are given as a proportion of columns by intestinal caeca and excretory vesicle. body length. Uterus short, passes from level of ovary to just anterior Schistorchis Sphincteristomum to ventral sucker. Excretory vesicle I-shaped, termi- skrjabini acollum nates at level of posterior margin of most posterior Locality Moreton Bay Lizard Island n 11 1 testis. Excretory arms extensive, to anterior margin of Host T. angustifrons A. stellatus oral sucker. Excretory pore terminal. Body L 1315–3651 (1959) 2126 Remarks Body W 456–789 (624) 1182 Body W % 21.6–41.4 (33.2) 55.6 This species was described by Parukhin (1963) from Forebody 307–627 (399) 554 the South China Sea in the triacanthid T. biaculeatus Forebody % 17.2–24.4 (21.1) 26.1 (as T. brevirostris) and from the balistid Abalistes Pre-oral L 29–78 (44) 57 stellatus (Bloch & Schneider) [as Abalistes stellaris OS L 211–369 (272) 385 (Bloch & Schneider)]; T. brevirostris was the first- OS W 217–368 (278) 418 named host and is interpreted as the type-host here. OS L % 10.1–17.2 (14.5) 18.1 The species was subsequently reported by Hafeezullah Prepharynx - 59 (1981) in T. biaculeatus (again as T. brevirostris) from Pharynx L 41–106 (68) 118 the Bay of Bengal. Pharynx W 69–172 (115) 200 The specimens reported here are strongly consistent VS L 101–194 (130) 168 with the two previous descriptions of Sch. skrjabini. VS W 94–179 (130) 171 The specimens also resemble Sch. paruchini Kuroch- VS L / OS L 0.42–0.53 (0.48) 0.44 kin, 1974 from a monacanthid, Meuschenia australis VS W / OS W 0.40–0.49 (0.47) 0.41 (Donovan) [as Navodon australis (Donovan)], from VS to ant. contiguous 107 the Great Australian Bight (Kurochkin, 1974). How- testis ever, Sch. paruchini differs from Sch. skrjabini in Testes no. 10–11 (10.9) 2 having a relatively larger ovary and vitelline follicles Ant. testis L 58–215 (130) 162 and infecting a monacanthid (vs mainly triacanthids). Ant. testis W 65–186 (120) 426 The report of this species in a balistid is probably Post. testis L - 224 exceptional or erroneous. In Moreton Bay, Sch. Post. testis W - 328 skrjabini occurs commonly in T. angustifrons but in Posttest. L 541–1899 (870) 555 none of multiple monacanthids and tetraodontids Posttest. L % 38.5–52.0 (43.8) 26.1 examined there. However, we have examined no Ovary L 79–202 (123) 134 balistids from this location. Ovary W 93–215 (141) 178 Vitelline field 744–2924 (1473) 1618 Sphincteristomum Oshmarin, Mamaev & Parukhin, L 1961 Previtelline L 299–586 (390) 458 Type-species: Sphincteristomum acollum Oshmarin, Postvitelline L 8–40 (27.5) 50 Mamaev & Parukhin, 1961 by original designation. Egg L 45–62 (55) 67 Egg W 25–36 (31) 43 Diagnosis. With characters of Schistorchiinae sensu Blend et al. (2017). Genus presently morphologically cryptic rel- of ventral sucker or just entering forebody to close to ative to Lobatotrema but phylogenetically distinct. posterior extremity, restricted to lateral margins Body ovate. Tegument spinous. Eye-spot pigment anteriorly, occupying most available space posterior dispersed in forebody. Pre-oral lobe present. Oral to testes, dorsally confluent immediately posterior to sucker highly glandular, pyriform; U-shaped partial 123 Syst Parasitol (2023) 100:381–413 393 Fig. 3 Relationships between species of the Apocreadiidae based on maximum likelihood phylogenetic analysis of the 28S dataset. Maximum likelihood bootstrap support values are shown above the nodes and Bayesian inference posterior probabilities shown below; values of\85 and\0.85 not shown. The scale-bar indicates expected number of substitutions per site. cox1 genotypes for P. stenosoma sequences shown. Abbreviations: G1, genotype 1; G2, genotype 2; LI, Lizard Island; Megap., Megaperinae. sphincter at aperture prominent. Ventral sucker round two, distinctly lobed, in anterior hindbody. Ovary in outline, smaller than oral sucker. Oesophagus entire, dextral or almost median in anterior hindbody, absent. Intestinal bifurcation immediately posterior contiguous with anterior testis. Vitelline follicles to pharynx, anterior to ventral sucker. Intestinal caeca distributed from near posterior extremity to level of open via separate ani at posterior end of body. Testes posterior margin of oral sucker, confluent in post- 123 394 Syst Parasitol (2023) 100:381–413 Table 4 Measurements of Lobatotrema aniferum. Percentages are given as a proportion of body length. Locality Lizard Island Lizard Island New Caledonia Ningaloo Reef n 8 3 3 10 Host Balistoides viridescens Pseudobalistes flavimarginatus Pseudobalistes fuscus Pseudobalistes fuscus Body L 1174–1412 (1250) 1674–1756 (1705) 1319–2151 (1802) 2120–3317 (2574) Body W 393–489 (446) 497–566 (540) 462–713 (610) 755–1240 (971) Body W % 33.1–39.1 (35.7) 28.3–33.8 (31.7) 33.1–35.6 (34.0) 30.8–42.2 (37.9) Forebody 326–413 (358) 413–441 (423) 414–538 (462) 552–708 (622) Forebody % 26.1–31.3 (28.6) 24.8–26.3 (24.8) 24.3–26.5 (25.2) 19.8– 29.2 (24.4) Pre-oral L 19–47 (32) 35–42 (39) 38–60 (48) 61–97 (75) OS L 195–222 (210) 212–262 (237) 245–296 (266) 268–398 (343) OS W 161–183 (170) 183–223 (202) 174–255 (220) 242–397 (326) OS L % 15.6–18.6 (16.8) 12.7–15.5 (13.9) 13.1–16.0 (14.3%) 10.9–16.5 (13.4) Prepharynx L 19–30 (24) 25–29 (27) - - Pharynx L 56–72 (63) 71–77 (74) 66–91 (78) 81–137 (109) Pharynx W 88–99 (94) 96–116 (106) 108–149 (128) 144–211 (186) VS L 82–112 (99) 96–125 (110) 98–142 (119) 140–212 (180) VS W 96–121 (108) 106–147 (123) 107–166 (138) 151–251 (209) VS L / OS L 0.41–0.53 (0.47) 0.45–0.48 (0.46) 0.43–0.48 (0.46) 0.47–0.56 (0.53) VS W / OS W 0.58–0.71 (0.64) 0.53–0.66 (0.61) 0.61–0.65 (0.63) 0.61–0.71 (0.64) VS to ant. testis 36–83 (65) 68–82 (76) 57–107 (82) 88164 (123) Testis no. 2 2 2 2 Ant. testis L 79–132 (110) 114–144 (130) 106–154 (130) 185–317 (230) Ant. testis W 128–162 (147) 203–231 (220) 173–277 (215) 338–522 (416) Post. testis L 102–150 (119) 159–208 (175) 130–226 (173) 268–407 (316) Post. testis W 106–152 (130) 177–215 (191) 164–251 (204) 305–469 (380) Posttest. L 436–606 (517) 760–852 (792) 525–1064 (830) 855–1600 (1108) Posttest. L % 37.0–43.9 (41.3) 45.1–48.5 (46.4) 39.8–49.5 (45.3) 35.9–49.0 (42.7) Ovary L 50–70 (61) 70–94 (81) 83–126 (99) 112–142 (128) Ovary W 69–84 (78) 109–141 (122) 95–164 (124) 164–227 (198) Vitelline field L 810–1054 (914) 1284–1316 (1305) 944–1666 (1367) 1660–2768 (2051) Previtelline L 270–377 (331) 378–422 (397) 387–477 (425) 438–683 (545) Postvitelline L 9–17 (13) 21–50 (32) 26–84 (42) 14–91 (48) Egg L 60 66–66 (66) 67–71 (69) 67–79 (72) Egg W 31 32–32 (32) 32–40 (36) 32–40 (36) testicular region. Excretory vesicle I-shaped, termi- Sphincteristomum acollum Oshmarin, Mamaev & nates close to posterior margin of posterior testis. Parukhin, 1961 Excretory pore terminal. In intestine of balistid fishes (Abalistes) in the Indo-Pacific and sparid fishes in the Type-host: Abalistes stellatus (Bloch & Schneider), Mediterranean Sea. Starry triggerfish (Tetraodontiformes: Balistidae). Type-locality: Tonkin Bay, Vietnam. Type-species: Sphincteristomum acollum Oshmarin, Mamaev & Parukhin, 1961. New material Other species: Sphincteristomum mediterraneae Host: A. stellatus. Abid-Kachour, Mouffok & Boutiba, 2013; Sphincter- Locality: off Lizard Island (1440’S, 14527’E), istomum nikolevi Parukhin, 1970. northern GBR, Australia. 123 Syst Parasitol (2023) 100:381–413 395 Fig. 4 Species of Schistorchiinae from fishes of Queensland. A, Schistorchis skrjabini Parukhin, 1963 from Tripodichthys angustifrons from Moreton Bay, south-eastern Queensland; B, Sphincteristomum acollum Oshmarin, Mamaev & Parukhin, 1961 from Abalistes stellatus off Lizard Island, northern GBR; C, Lobatotrema aniferum Manter, 1963 from Balistoides viridescens off Lizard Island. Scale- bars, A, B, 500 lm; C, 250 lm. Site in host: Intestine. Description (Fig. 4B) Prevalence: 1 of 1 (100%). Deposition of specimens: Hologenophore QM [Based on a single whole-mounted hologenophore G240423. from A. stellatus from off Lizard Island.] Body broad, Representative DNA sequences: Partial cox1 mtDNA, with maximum breadth in mid-hindbody; posterior one sequence (submitted to GenBank, OQ445530); margin damaged. Tegument spinous to mid-hindbody. ITS2 rDNA, one sequence (submitted to GenBank, Pre-oral lobe distinct. Eye-spot pigment dispersed in OQ442920); partial 28S rDNA, one sequence (sub- forebody. Oral sucker roughly pyriform, highly glan- mitted to GenBank, OQ442907). dular; U-shaped muscular sphincter embedded at Measurements: Table 3. aperture. Prepharynx short. Pharynx large, rounded, with anterior portion dorsal to oral sucker. Oesopha- gus absent. Intestinal bifurcation just anterior to anterior margin of ventral sucker. One caecum open- ing at anus on damaged posterior margin; end of 123 396 Syst Parasitol (2023) 100:381–413 second caecum removed in portion used for sequenc- 1200 ing. Ventral sucker round in outline, unspecialised, far Body width 1000 smaller than oral sucker. Testes two, tandem, con- tiguous, in anterior hindbody, distinctly lobed; ante- 800 rior testis significantly wider than long; posterior testis 600 triangular; post-testicular region occupying approxi- 400 mately two-fifths body length (based on estimated full body length). Seminal vesicle saccular, prominent, 1000 2000 3000 Body length dextral to ventral sucker. Genital pore ventro-subme- dian, slightly sinistral, immediately anterior to ventral 250 Ventral sucker width sucker. Ovary ovoid, median, contiguous with anterior testis, mostly posterior to ventral sucker. Canalicular 200 seminal receptacle not detected. Vitelline follicles extend from level of posterior margin of oral sucker to 150 close to posterior extremity, mainly restricted to 100 lateral margins anteriorly, occupying most available space around and posterior to testes, dorsally confluent 200 300 400 500 immediately posterior to testes then broadly separated Oral sucker width into columns by intestinal caeca and excretory vesicle. Uterus short, between level of ovary and genital pore. Excretory vesicle I-shaped, reaches to posterior testis. Body length 3000 Excretory pore missing from damaged posterior extremity. 2000 Remarks 1000 This species was described from A. stellatus (as 1 2 3 4 5 6 7 Host/locality combination Abalistes stellaris) from off Vietnam by Oshmarin et al. (1961) with two subsequent reports from the Fig. 5 Morphometric comparisons of Lobatotrema aniferum South China Sea (Parukhin & Chikunova, 1964; Manter, 1963. A, Body length vs body width for Sch. acollum (d Parukhin, 1989), neither of which included a descrip- from original description by Oshmarin et al. (1961); j from tion or figure. Manter (1963) reported Lobatotrema Abalistes stellatus¸ this study) and L. aniferum (  from description of L. aniferum from unknown balistid from Fiji by aniferum from an unidentified balistid from Fiji with Manter (1963); h from B. viridescens from Japan; 4 from P. the species being synonymised with Sph. acollum by fuscus from Japan; 5 from B. viridescens from Lizard island; e Yamaguti (1971). Machida & Kuramochi (1999) from P. flavimarginatus from Lizard island;  from P. fuscus reported Sph. acollum from two other balistids, from Ningaloo Reef; w from P. fuscus from New Caledonia. B, Oral sucker width vs ventral sucker width for Sch. acollum ( d Balistoides viridescens (Bloch & Schneider) and from original description by Oshmarin et al. (1961); j from A. Pseudobalistes fuscus (Bloch & Schneider), from off stellatus¸ this study) and L. aniferum (w from description of L. Japan. As discussed further following the report of the aniferum from unknown balistid from Fiji by Manter (1963);  species, we consider L. aniferum valid and that the from B. viridescens and P. fuscus from Japan; m all other specimens from Ningaloo Reef, the GBR and New Caledonia. Japanese specimens should be identified as L. ani- C, Lengths of specimens of L. aniferum (1, B. viridescens, ferum rather than Sph. acollum. In our view, Sph. Lizard Island; 2, P. flavimarginatus, Lizard Island; 3, P. fuscus, acollum has only been reported from A. stellatus. New Caledonia; 4, P. fuscus, Japan; 5, unknown balistid, Fiji; 6, The single hologenophore reported here was P. fuscus, Ningaloo Reef; 7, B. viridescens, Japan). incomplete at the time of collection, missing part of its posterior end, possibly from attack by other report constitutes a new host and locality record for helminths. Apart from this damage, it is strongly this species and the first record of it from Australia. consistent with the original species description. This 123 Syst Parasitol (2023) 100:381–413 397 Lobatotrema Manter, 1963 Prevalence: off Lizard Island: 1 of 2 (50%) B. Type-species: Lobatotrema aniferum Manter, 1963 viridescens; 1 of 1 P. flavimarginatus. Ningaloo Reef: by original designation. 4 of 7 (57%) P. fuscus. New Caledonia 1 of 1 P. fuscus. Deposition of specimens: QM G240424–38; WAM Diagnosis. V11697–712; MNHN HEL1900–4. Representative DNA sequences: Partial cox1 mtDNA, With characters of Schistorchiinae sensu Blend et al. eight sequences (three submitted to GenBank, (2017). Genus presently morphologically cryptic rel- OQ445531–33); ITS2 rDNA, 11 sequences (three ative to Sphincteristomum but phylogenetically dis- submitted to GenBank, OQ442921–23); partial 28S tinct. Body elongate to elliptical. Tegument spinous. rDNA, three sequences (two submitted to GenBank, Eye-spot pigment dispersed in forebody. Pre-oral lobe OQ442908–09). present. Oral sucker highly glandular, pyriform; Measurements: Table 4. U-shaped partial sphincter at aperture prominent. Ventral sucker round in outline, smaller than oral Description (Fig. 4C) sucker. Oesophagus absent. Intestinal bifurcation immediately posterior to pharynx, anterior to ventral [Based on eight wholemount specimens, including sucker. Intestinal caeca open via separate ani at two hologenophores from B. viridescens from off posterior end of body. Testes two, entire to distinctly Lizard Island.] Body elongate linguiform, with max- lobed, in anterior hindbody. Ovary entire, dextral in imum breadth at level of testes; posterior end slightly anterior hindbody, contiguous with anteriormost tapered, bluntly rounded. Tegument spinous in fore- testes. Vitelline follicles distributed from near poste- body. Pre-oral lobe distinct. Eye-spot pigment dis- rior extremity to posterior forebody, confluent in post- persed in forebody. Oral sucker ovoid to elongate testicular region. Excretory vesicle I-shaped, termi- pyriform, highly glandular; U-shaped muscular nates close to posterior margin of posterior testis. sphincter embedded at aperture. Prepharynx short. Excretory pore terminal. In intestine of balistid fishes Pharynx subquadrate, immediately posterior to or (Balistoides, Pseudobalistes) in the Indo-Pacific. slightly overlapping oral sucker. Oesophagus insignif- Type- and only species: Lobatotrema aniferum Man- icant to absent. Intestinal bifurcation close to level of ter, 1963. anterior margin of ventral sucker. Caeca open sepa- rately at posterior extremity at marginal ani. Ventral Lobatotrema aniferum Manter, 1963 sucker round in outline, unspecialised, far smaller than Syn: Sphincteristomum acollum of Yamaguti (1971) oral sucker. Testes two, tandem, in midbody, contigu- in part and of Machida and Kuramochi (1999). ous, usually entire, sometimes distinctly lobed; post- Type-host: ‘‘Balistid sp.’’ (Tetraodontiformes: testicular region occupying approximately two-fifths Balistidae) body length. Seminal vesicle saccular, small, dextral, Type-locality: Off Fiji. mainly dorsal to ventral sucker. Genital pore imme- diately anterior to ventral sucker, slightly sinistrally New material submedian. Ovary ovoid, slightly dextral, contiguous Hosts: Balistoides viridescens (Bloch & Schneider), with anterior testis, usually immediately posterior to Titan triggerfish; Pseudobalistes flavimarginatus ventral sucker. Canalicular seminal receptacle saccu- (Rüppell), Yellowmargin triggerfish; Pseudobalistes lar, dorsal to ovary. Vitelline follicles extend from just fuscus (Bloch & Schneider), Yellow-spotted trigger- anterior to ventral sucker, usually paralleling intestinal fish (Tetraodontiformes: Balistidae). caeca, to close to posterior extremity, mainly restricted Localities: off Lizard Island (1440’S, 14527’E), to lateral margins anteriorly, becoming more extensive northern GBR; Ningaloo Reef (2155’S, 11358’E), posteriorly and occupying most available space Western Australia, Australia. Off Îlot Goéland around and posterior to testes, dorsally confluent (2222’S, 16622’E), New Caledonia. immediately posterior to testes then broadly separated Site in host: Intestine. into four poorly defined columns by intestinal caeca and excretory vesicle. Uterus extends anteriorly from level of posterior margin of ovary. Excretory vesicle 123 398 Syst Parasitol (2023) 100:381–413 I-shaped, reaches to posterior testis. Excretory pore reasonably clear. Charting of the widths of the oral terminal. and ventral suckers (Fig. 5B) suggests three groups of specimens – the two Sph. acollum, Japanese speci- Remarks mens, and those from Ningaloo Reef, the GBR, New Caledonia, and Fiji. Lobatotrema aniferum was described by Manter In summary, for specimens that we identify as (1963) from an unidentified balistid from Fiji. The representing Sph. acollum and L. aniferum we find species was synonymised with Sph. acollum by unambiguous molecular distinction together with Yamaguti (1971) following a personal communication limited distinction based on morphology and host with Manter who evidently agreed with the synonymy. distribution. One morphological character suggests a The two species are highly similar, and we were first possible distinction between Japanese specimens of L. alerted to their apparent distinction by finding that aniferum and those from elsewhere, but we hesitate to relevant sequence data are unambiguously consistent identify them as distinct in the absence of any other with the presence of two species and, potentially, in evidence (molecular data or distinction in host). terms of phylogenetic relationships, two genera. Notably, specimens from Japan were the largest Overall, the combined morphological, molecular and examined (Fig. 5C), but specimens from Ningaloo host distribution evidence shows decisively that the Reef were almost as large. In the cox1 dataset, new specimens reported here are distinct from Sph. specimens from Ningaloo Reef and off Lizard Island acollum and consistent with the original description of form two distinct lineages, differing by 22 bp; ITS2 L. aniferum by Manter (1963). Machida & Kuramochi sequences for samples from the two localities differ at (1999) reported specimens of Sph. acollum from Japan 1 bp and 28S sequences were identical. These from two of the host species reported here. Although molecular data are best interpreted as relating to there are no supporting molecular data available, geographical variation of a single species which may morphology and host distribution lead us to conclude involve regional variation in overall size. In our view, that the Japanese specimens are best identified as L. the evidence suggests that Lobatotrema is best aniferum. considered a ‘‘cryptic genus’’; it is phylogenetically The clearest indication of the distinction between distinct but morphologically undifferentiated from the Sph. acollum and L. aniferum is in the sequences concept of Schistorchis. Whether it is useful to generated from different balistid species in sympatry. recognise such taxa is a complex issue; recognising cox1, ITS2 and 28S sequence data for these two cryptic genera may reflect biological reality whereas species differ at 91–98, 18–19 and 65 bp respectively requiring morphological distinction maintains taxo- and, in phylogenetic analysis, the two species do not nomic utility. We find a small literature considering form a clade. In contrast, we found no reliable genus- cryptic genera (Hsieh et al., 2014; Maggioi et al., level morphological distinctions. We considered the 2018; Lehr et al., 2020), especially for Cyanobacteria form of the oral sphincter, described originally as (e.g. Shalygin et al., 2017; Pietrasiak et al., 2021) U-shaped for Sph. acollum and V-shaped for L. which might be predicted to be problematic given their aniferum, but found no fundamental distinction in limited morphological variability. Here we propose to the specimens that we examined. The original descrip- recognise Lobatotrema because the available evidence tion of Sph. acollum and our figured hologenophore suggests that it is real, an available name already exists specimen both have somewhat lobed testes whereas for it, and to draw attention to what we suspect is a that in our figure of L. aniferum has smooth testes, but developing problem in trematode taxonomy as the some of our specimens of L. aniferum have testes that molecular database expands for taxa with limited are distinctly lobed, although perhaps not as strongly morphological variability (e.g. Yong et al., 2021). The as in Sph. acollum. From morphometric analysis we generic diagnosis proposed above differs from that of found two distinctions between the two species. The Sphincteristomum only in the identified host range and body of specimens of Sph. acollum is relatively in specific reference to phylogenetic distinction. broader than that of L. aniferum (Fig. 5A); one of 38 Relative to the two other species of Sphincteristo- Japanese specimens approaches the condition of Sph. mum, L. aniferum is easily distinguished from Sph. acollum but otherwise the distinction seems nikolevi Parukhin, 1970 (from a balistid, Rhinecanthus 123 Syst Parasitol (2023) 100:381–413 399 Table 5 Measurements of two new species of Blendiella. Percentages are given as a proportion of body length. B. tridecimtestis B. trigintatestis Locality Lizard Island Lizard Island Lizard Island Lizard Island n 19 8 12 7 Host Balistapus undulatus Balistapus undulatus Sufflamen chrysopterum Rhinecanthus aculeatus Body L 1003–1397 (1234) 816–1631 (1258) 1227–1566 (1374) 816–1273 (1057) Body W 210–335 (267) 214–343 (283) 237–311 (277) 214–290 (257) Body W % 17.1–26.5 (21.7) 19.9–26.2 (22.8) 18.0–23.6 (20.2) 21.4–28.3 (24.5) Forebody 187–253 (219) 191–273 (237) 203–248 (222) 191–246 (214) Forebody % 15.7–19.3 (17.8) 15.8–23.4 (19.3) 15.1–17.8 (16.2) 16.7–23.4 (20.5) Pre-oral L 15–37 (23) 25–42 (32) 19–29 (25) 18–24 (21) OS L 108–137 (118) 96–126 (111) 101–117 (111) 96–117 (106) OS W 117–162 (136) 112–163 (134) 115–139 (128) 112–133 (121) OS L % 8.2–11.2 (9.6) 7.4–11.8 (9.1) 7.3–8.9 (8.1) 8.7–12.3 (10.2) Prepharynx L 12–22 (16) 10–18 (14) 11–25 (14) 12–16 (14) Pharynx L 28–46 (38) 29–39 (35) 27–45 (34) 28–34 (32) Pharynx W 54–80 (64) 50–72 (60) 51–66 (60) 52–67 (59) VS L 60–83 (71) 69–89 (76) 66–97 (78) 64–77 (70) VS W 60–83 (74) 69–95 (81) 71–105 (84) 69–85 (76) VS L / OS L 0.52–0.68 (0.60) 0.63–0.75 (0.68) 0.57–0.94 (0.71) 0.61–0.72 (0.67) VS W / OS W 0.45–0.64 (0.60) 0.55–0.66 (0.60) 0.55–0.82 (0.66) 0.58–0.70 (0.63) VS to ant. testis 48–98 (66) 46–101 (70) 67–104 (87) 50–81 (71) Testis no. 10–16 (13) 28–32 (29) 25–32 (29) 25–33 (29) Ant. testis L 22–42 (33) 28–45 (37) 26–47 (38) 28–44 (37) Ant. testis W 25–48 (37) 26–50 (42) 32–58 (43) 41–48 (45) Posttest. L 414–651 (502) 120–518 (301) 234–412 (325) 120–300 (202) Posttest. L % 34.4–48.9 (40.6) 11.1–31.8 (23.0) 16.8–30.5 (23.8) 11.1–28.3 (19.5) Ovary L 51–91 (71) 49–78 (61) 61–93 (81) 47–73 (58) Ovary W 47–82 (68) 54–83 (65) 64–95 (75) 54–82 (62) Vitelline field L 821–1196 (1024) 623–1391 (1023) 982–1331 (1140) 623–1039 (832) Previtelline L 171–232 (204) 187–283 (246) 202–254 (226) 187–266 (226) Postvitelline L 10–23 (16) 10–26 (19) 10–20 (14) 12–20 (15) Egg L 47–65 (55) 50–66 (60) 52–70 (61) 57 Egg W 29–44 (34) 28–38 (33) 26–43 (33) 36 sp., from the Red Sea), which has opposite testes restricted to species of Balistoides Fraser-Brunner and (Parukhin, 1970) and from Sph. mediterraneae [from a Pseudobalistes Bleeker; unfortunately, the type-host sparid, Pagellus erythrinus (Linnaeus) from the of the species in Fiji was not identified beyond family. Mediterranean], which has the testes at the posterior McCord & Westneat (2016) inferred phylogenetic end of the body (Abid-Kachour et al., 2013). Sphinc- relationships of the Balistidae, recognising three teristomum mediterraneae is exceptional both in being clades. Of the species of interest here, P. fuscus fell reported from a sparid and in being the only in Clade 1, B. viridescens and P. flavimarginatus fell schistorchiine species reported from outside of the as sister taxa in Clade 2, and the species of Abalistes Indo-west Pacific. (host of Sph. acollum) fell in Clade 3. These results The evidence from New Caledonia, Ningaloo Reef, indicated that neither Balistoides nor Pseudobalistes the GBR and Japan, suggests that L. aniferum is as presently constituted is monophyletic. The 123 400 Syst Parasitol (2023) 100:381–413 proposed solution included recognition of P. fuscus as Remarks a species of Balistes and B. viridescens as a species of Pseudobalistes. These changes do not appear to have In the recent classification proposed by Blend et al. been adopted in the fish literature, although we detect (2017), the two new species described below would no literature presenting a dissenting view. Regardless, fall unambiguously in Schistorchis on the basis of their the balistid relationships reported by McCord & highly glandular oral suckers and large numbers of Westneat (2016) have the hosts of L. aniferum in testes. However, the molecular phylogenetic analyses two (non-sister) clades and those of Sph. acollum in presented here show that the two new species form a the third. Unfortunately, these insights do not mate- clade distant from the type-species of Schistorchis, rially improve understanding of genus-level-distinc- Sch. carneus, which forms a strongly supported clade tion between Lobatotrema and Sphincteristomum. with one other sequenced species consistent with the present concept for the genus, Sch. skrjabini. If Blendiella n. gen. schistorchiine genera are to be maintained as mono- phyletic, several solutions to this problem are possible. Diagnosis. Schistorchis and Sphincteristomum could be syn- onymised, recognising Schistorchis as a genus in which the testis number ranges from 2–33. We find With characters of Schistorchiinae sensu Blend et al. that idea unsatisfactory for these parasites, although (2017). Body relatively small, elongate with almost we note that Miller & Cribb (2013) took that approach parallel sides. Tegument spinous. Eye-spot pigment in recognising Siphomutabilis Miller & Cribb, 2013 dispersed in forebody. Pre-oral lobe present. Oral for cryptogonimids with two or multiple testes. sucker highly glandular, pyriform; U-shaped partial Megacreadium Nagaty, 1956, presently a synonym sphincter at aperture prominent. Ventral sucker round of Schistorchis, could be re-recognised, but we see no in outline, smaller than oral sucker. Oesophagus basis to consider its poorly known type species, M. absent. Intestinal bifurcation immediately posterior tetraodontis Nagaty, 1956, distinct from Schistorchis to pharynx, anterior to ventral sucker. Intestinal caeca sensu stricto or as consistent with the two new species. open via separate ani at posterior end of body. Testes Instead, we propose a new genus on the basis that the 10–33 (rarely\12), entire, in single column up to four species included are not monophyletic with Sch. testes wide anteriorly, usually narrowing to single carneus and because we recognise some narrow testis wide posteriorly. Post-testicular region varying morphological differences as follows. The two new strongly in proportion with number of testes. Ovary species have 25–33 (29) (B. trigintatestis) and 10–16 entire, dextral in anterior hindbody, contiguous with (13) (B. tridecimtestis) testes whereas species of anterior testis. Vitelline follicles distributed from near Schistorchis almost always have fewer (cf. Sch. posterior extremity to anterior margin of ventral carneus – 11 in all reports; Sch. paruchini – 11 in all sucker or into forebody, confluent in post-testicular reports; Sch. skrjabini – 11 in all reports; Sch. region. Excretory vesicle I-shaped, terminates anteri- tetraodontis – 8 in single reported specimen). Just orly well posterior to posteriormost testis. Excretory two of 19 B. tridecimtestis had fewer than 12 testes. pore terminal. In intestine of balistid fishes in the The two new species are also significantly smaller and Pacific Ocean. narrower than the four Schistorchis species. The Type-species: Blendiella trigintatestis n. sp. largest specimen of the new genus is 1,631 lm long Other species: Blendiella tridecimtestis n. sp. in comparison with recorded maxima of 14,400 lm for Etymology: The new name honours our colleague Sch. carneus, 2,900 lm for S. kurochkini, 3,600 lm for Professor Charles (Chuck) Blend and his extended S. skrjabini and 13,500 lm for Sch. tetraodontis. The contribution to this field, including specifically to the oral suckers of the two new species have a distinctive understanding of the Schistorchiinae. shape, constricted anteriorly, whereas those of species ZooBank LSID: urn:lsid:zoobank.org:act:3F9CF5AA- of Schistorchis are evenly round in outline. Finally, the DCB0-4F70-AD47-51E865EEF228. two new species both infect balistids whereas the four recognised species of Schistorchis are reported from monacanthids, tetraodontids and triacanthids. We 123 Syst Parasitol (2023) 100:381–413 401 Deposition of specimens: QM G240439–72. Representative DNA sequences: Partial cox1 mtDNA, six sequences (two submitted to GenBank, OQ445534–35); ITS2 rDNA, nine sequences (three submitted to GenBank, OQ442924–26); partial 28S rDNA, two sequences (one submitted to GenBank, OQ442911). Measurements: Table 5. ZooBank LSID: urn:lsid:zoobank.org:act:86F228BA- 4F4F-4FEF-B082-3CD8A82540E8. Etymology: the specific name is composed from the Latin for 30 (triginta), and testis, in reference to the exceptional number of testes in this species. Description (Fig. 6A) [Based on 14 wholemount specimens, including one hologenophore from S. chrysopterum from off Lizard Island.] Body small, elongate with nearly parallel margins, maximum breadth anywhere from level of ventral sucker to mid-hindbody; posterior end tapered and bluntly rounded. Tegument spinous in anterior forebody. Pre-oral lobe distinct. Eye-spot pigment dispersed in forebody. Oral sucker pyriform, with broad posterior margin, highly glandular; small U-shaped muscular sphincter embedded at aperture. Prepharynx short. Pharynx subquadrate, muscular and Fig. 6 Species of Blendiella n. gen. from fishes off Lizard immediately posterior to oral sucker, sometimes Island, northern Great Barrier Reef. A, Blendiella trigintatestis dorsally overlapping oral sucker slightly. Oesophagus n. sp. from Sufflamen chrysopterum; B, Blendiella tridecimtestis absent. Intestinal bifurcation in mid-forebody. Caeca n. sp. from Sufflamen chrysopterum. Scale-bars, A, B, 250 lm. open at separate marginal ani close to posterior extremity. Ventral sucker round in outline, unspe- acknowledge that these distinctions are relatively cialised, far smaller than oral sucker. Testes 25–33, subtle, but in combination they allow recognition of entire, contiguous, arranged in broad column from just the new genus. posterior to ventral sucker to one-third of hindbody length from posterior extremity; column initially three Blendiella trigintatestis n. sp. or four testes wide, but narrows progressively, even- Type-host: Sufflamen chrysopterum (Bloch & Schnei- tually to one posteriorly. Seminal vesicle small, der), Halfmoon triggerfish (Tetraodontiformes: saccular, dorso-dextral to ventral sucker. Genital pore Balistidae). immediately anterior to ventral sucker, slightly sin- Other hosts: Rhinecanthus aculeatus (Linnaeus), istro-submedian. Ovary roughly ovoid, posterior, and White-banded triggerfish; Balistapus undulatus slightly dextral to ventral sucker, contiguous with (Park), Orange-lined triggerfish (Tetraodontiformes: anteriormost dextral testes. Canalicular seminal recep- Balistidae). tacle saccular, antero-dorsal to ovary. Vitelline folli- Type-locality: off Lizard Island (1440’S, 14527’E), cles extend from just anterior to ventral sucker to close northern GBR, Australia. to posterior extremity, mainly paralleling intestinal Site in host: Intestine. caeca anteriorly, becoming more extensive posteriorly Prevalence: 37 of 78 (47%) S. chrysopterum; 7 of 39 and occupying most available space around and (18%) R. aculeatus; 13 of 19 (68%) B. undulatus. posterior to testes, confluent immediately posterior 123 402 Syst Parasitol (2023) 100:381–413 to testes then broadly separated into four poorly Table 6 Measurements of Paraschistorchis stenosoma. Per- defined columns by intestinal caeca and excretory centages are given as a proportion of body length. vesicle. Uterus short, restricted to area between ovary Locality Heron Island Heron Island and pharynx. Excretory vesicle I-shaped, relatively n 18 13 short, terminates well short of posterior testis. Excre- Host Cantherhines Cantheschenia tory pore terminal. pardalis grandisquamis Body L 1629–3448 1337–2875 (2083) Remarks (2432) Body W 267–565 (421) 312–548 (411) This species is unique within the concept of the Body W % 13.8–22 (18.2) 17.1–23.3 (20) Schistorchiinae in the high number of testes (25–33). Forebody 306–536 (426) 262–522 (376) For both this and the following species, many Forebody % 14.6–22.7 (18.3) 16.3–22.6 (18.3) specimens were damaged, perhaps by partial predation Pre-oral L 19–45 (35) 22–55 (36) by other helminths. In these incomplete specimens the OS L 157–245 (204) 138–212 (177) testis number may be dramatically reduced, but the OS W 158–251 (214) 146–221 (181) identity of the specimen is still usually recognisable by OS L % 6.6–10.6 (8.8) 7.4–10.3 (8.7) the organisation of the remaining testes. Prepharynx L 23–46 (33) 19–37 (28) Pharynx L 62–123 (94) 58–104 (81) Blendiella tridecimtestis n. sp. Pharynx W 68–157 (124) 90–154 (113) Type-host: Balistapus undulatus (Park), Orange-lined VS L 115–461 (176) 116–189 (149) triggerfish (Tetraodontiformes: Balistidae). VS W 128–544 (189) 121–204 (161) Other host: Sufflamen chrysopterum (Bloch & Sch- VS L / OS L 0.71–0.84 (0.78) 0.76–0.90 (0.84) neider), Halfmoon triggerfish (Tetraodontiformes: Balistidae). VS W / OS W 0.70–0.86 (0.78) 0.82–0.94 (0.89) Type-locality: off Lizard Island (1440’S, 14527’E), VS to ant. 117–449 (297) 139–329 (227) testis L northern GBR, Australia. Testis no. 10–11 (11) 11–11 (11) Site in host: Intestine. Ant. testis L 52–142 (79) 59–134 (92) Prevalence: 8 of 19 (42.1%) B. undulatus; 1 of 78 Ant. testis W 75–166 (104) 95–191 (134) (1.2%) S. chrysopterum. Posttest. field 59–1139 (627) 311–829 (598) Deposition of specimens: QM G240473–97. L Representative DNA sequences: Partial cox1 mtDNA, Posttest. field 15.6–33.0 (27.5) 23.3–28.8 (28.7) six sequences (five submitted to GenBank, L% OQ445536–40); ITS2 rDNA, five sequences (two Ovary L 70–176 (125) 84–152 (113) submitted to GenBank, OQ442927–28); partial 28S Ovary W 80–178 (131) 85–145 (120) rDNA, two sequences (one submitted to GenBank, Vitelline field 905–2361 (1590) 805–1944 (1403) OQ442910). L Measurements: Table 5. Previtelline L 485–1038 (772) 457–909 (669) ZooBank LSID: urn:lsid:zoobank.org:act:A6DB Postvitelline L 13–585–(109) 21–85 (55) 51BA-F5FC-4372-9ECF-C0F65B45C805. Egg L 52–69 (60) 55–68 (61) Etymology: the specific name is composed from the Egg W 30–39 (34) 30–37 (33) Latin for 13 (tridecim) and testis in reference to the most common number of testes in this species. from level of ventral sucker to mid-hindbody. Poste- Description (Fig. 6B) rior end tapered and bluntly rounded. Tegument spinous in anterior forebody. Pre-oral lobe distinct. [Based on 30 wholemount specimens, including five Eye-spot pigment dispersed in forebody. Oral sucker hologenophores from B. undulatus from off Lizard pyriform, with broad posterior margin, highly glan- Island]. Body relatively small, elongate with nearly dular; small U-shaped muscular sphincter embedded parallel margins, with maximum breadth anywhere at aperture. Prepharynx short. Pharynx subquadrate, 123 Syst Parasitol (2023) 100:381–413 403 testes. Canalicular seminal receptacle saccular, antero-dorsal to ovary. Vitelline follicles extend from about level of pharynx to close to posterior extremity, mainly paralleling intestinal caeca anteriorly, becom- ing more extensive posteriorly and occupying most available space around and posterior to testes, conflu- ent immediately posterior to testes then broadly separated into four columns by intestinal caeca and excretory vesicle. Uterus short, restricted to area between ovary and pharynx. Excretory vesicle I-shaped, relatively short, terminates well short of posterior testis. Excretory pore terminal. Remarks This species is easily distinguished from its congener by possessing only 11–16 (vs 25–33) testes. Relative to the four species of Schistorchis with which it shares multiple testes and a glandular oral sucker, this is a distinctively small and narrow species. In addition, the testis number is almost always slightly higher than the maximum of 11 seen in those species (overlapping rarely), and the testes are principally in a single column whereas in the four species of Schistorchis they are mainly distributed lateral to each other. This species is clearly closely related to B. trigin- tatestis with which it co-occurs in B. undulatus. Fig. 7 Species of Paraschistorchis from fishes of the Great Barrier Reef. A, Paraschistorchis stenosoma (Hanson, 1953) However, there is an apparent distinction in host- from Cantherhines pardalis off Heron Island; B, Paraschis- specificity in that this species has been found torchis seychellesiensis (Toman, 1989) from C. pardalis off overwhelmingly in B. undulatus whereas B. trigintat- Heron Island. Scale-bars, A, B, 500 lm. estis also occurs frequently in two other balistid species. muscular and immediately posterior to oral sucker, sometimes dorsally overlapping oral sucker slightly. Paraschistorchis Blend, Karar & Dronen, 2017 Oesophagus absent. Intestinal bifurcation in mid- Type-species: Paraschistorchis stenosoma (Hanson, forebody. Caeca open at separate marginal ani close 1953)Blend, Karar & Dronen, 2017 by original to posterior extremity. Ventral sucker round in outline, designation. unspecialised, far smaller than oral sucker. Testes 11–16, entire, contiguous anteriorly, slightly separated Diagnosis posteriorly, arranged in broad column from distinctly posterior to ventral sucker to mid-hindbody; column With characters of Schistorchiinae sensu Blend et al. initially two or three testes wide but rapidly narrows (2017). Body elongate to elliptical. Tegument spinous. progressively to one for posterior half. Seminal vesicle Eye-spot pigment dispersed in forebody. Pre-oral lobe small, saccular, dorso-dextral or dorso-posterior to present or inconspicuous. Oral sucker mainly muscu- ventral sucker. Genital pore immediately anterior to lar but some species with distinct glandular elements, ventral sucker, sightly sinistro-submedian. Ovary round in outline; U-shaped partial sphincter at aperture roughly ovoid, posterior to (sometimes contiguous prominent or almost undetectable. Ventral sucker with, sometimes slightly separated from) and slightly round in outline, smaller than oral sucker. Oesophagus dextral to ventral sucker, contiguous with anteriormost short but distinct. Intestinal bifurcation immediately 123 404 Syst Parasitol (2023) 100:381–413 anterior to ventral sucker. Intestinal caeca open via OQ445541–44); ITS2 rDNA, seven sequences (two separate ani at posterior end of body. Testes normally submitted to GenBank, OQ442929–30); partial 28S 11 (rarely fewer), entire, anteriorly in column one or rDNA, four sequences (two submitted to GenBank, two testes wide, posteriorly reduces to single testis. OQ442912–13). Ovary entire, dextral or almost median in anterior Measurements: Table 6. hindbody, contiguous with anterior testis. Vitelline follicles distributed from near posterior extremity to Description (Fig. 7A) anywhere from level of posterior margin of ovary to posterior forebody. Excretory vesicle I-shaped, termi- [Based on 18 wholemount specimens, including three nates close to posterior margin of posterior testis. hologenophores from C. pardalis from off Heron Excretory pore terminal. In intestine principally of Island.] Body narrow, with almost parallel margins monacanthid, siganid and zanclid fishes in the Pacific and maximum breadth usually slightly anterior to and Indian Oceans. ventral sucker. Posterior margin rounded, roughly Type-species: Paraschistorchis stenosoma (Han- symmetrical relative to anterior margin. Tegument son, 1953) Blend, Karar & Dronen, 2017. spinous to level of ventral sucker. Pre-oral lobe Other species: Paraschistorchis longivesiculurus distinct. Eye-spot pigment dispersed in forebody. Oral (Hafeezullah, 1981) Blend, Karar & Dronen, 2017; sucker rounded in outline, muscular but with clear Paraschistorchis seychellesiensis (Toman, 1989) glandular components; U-shaped muscular sphincter Blend, Karar & Dronen, 2017; P. zancli (Hanson, embedded at aperture discernible, but not conspicu- 1953) Blend, Karar & Dronen, 2017. ous. Prepharynx short. Pharynx subquadrate, typically partly dorsal to oral sucker. Oesophagus distinct. Paraschistorchis stenosoma (Hanson, 1953) Blend, Intestinal bifurcation immediately anterior to ventral Karar & Dronen, 2017 sucker. Caeca broad, terminate at separate ani at Syn. Schistorchis stenosoma Hanson, 1953 posterior extremity. Ventral sucker round in outline, Type-host: Cantherhines sandwichiensis (Quoy & unspecialised, only marginally smaller than oral Gaimard) Sandwich Isle file [reported as Cantherhines sucker. Testes usually 11, (6, 7, 9 in each of one pardalis (Rüppell), Honeycomb filefish] (Tetraodon- specimen), usually contiguous, commencing well tiformes: Monacanthidae). posterior to ventral sucker, arrangement dependent Type-locality: Hawaii, United States of America on extension of body; in well-extended specimens (USA). testes in single column; in more contracted specimens, testes may be opposite anteriorly and zigzag posteri- New material orly; post-testicular region occupying approximately Hosts: Cantherhines pardalis; Cantheschenia gran- one-quarter body length. Seminal vesicle saccular, disquamis Hutchins, Large-scaled leatherjacket small, postero-dorsal to ventral sucker. Genital pore (Tetraodontiformes: Monacanthidae). ventro-submedian, immediately anterior to ventral Localities: off Lizard Island (1440’S, 14527’E), sucker, slightly sinistral. Ovary rounded in outline, northern GBR; off Heron Island (2327’S, 15155’E), well-separated from and slightly dextral to ventral southern GBR, Australia. sucker. Canalicular seminal receptacle not detected. Site in host: Intestine. Vitelline follicles extend from about level of posterior Prevalence: off Lizard Island: 3 of 9 (33%) C. margin of ovary to close to posterior extremity, pardalis. Off Heron Island: 17 of 35 (49%) Canther- occupying most available space around and posterior hines pardalis; 26 of 43 (60%) Cantheschenia to testes, unevenly ventrally confluent posterior to grandisquamis. testes, only weakly separated into columns by intesti- Deposition of specimens: Hologenophores Mitochon- nal caeca and excretory vesicle. Uterus relatively long, drial Genotype A QM G240498–500; vouchers not extends slightly posterior to ovary, fills space between differentiated by mitochondrial sequences QM ovary and ventral sucker. Excretory vesicle I-shaped, G240501–27. reaches to posterior testis. Excretory pore terminal. Representative DNA sequences: Partial cox1 mtDNA, six sequences (four submitted to GenBank, 123 Syst Parasitol (2023) 100:381–413 405 Table 7 Measurements of Paraschistorchis seychellesiensis 1963; Yamaguti, 1970). We infer that the fish species and P. zancli. Percentages are given as a proportion of body involved was actually C. sandwichiensis, a close length. relative of C. pardalis which occurs commonly at P. seychellesiensis P. zancli Hawaii, as C. pardalis does not occur in that region Locality Heron Island, GBR Moorea, FP (Myers, 1991). Yamaguti (1970) also reported it from n 10 10 Host Cantherhines Zanclus cornutus Sufflamen fraenatum (Latreille) (Balistidae). pardalis Paraschistorchis presently comprises four species: P. longivesiculurus, P. seychellesiensis, P. stenosoma Body L 1120–4348 (2532) 1448–1973 and P. zancli. Among these, P. seychellesiensis is (1681) easily distinguished as the broadest species. Body W 315–1301 (891) 244–306 (272) Paraschistorchis longivesiculurus is distinctive in Body W % 16.2–39.6 (30.8) 15.5–16.9 (16.2) having the testes reach close to the posterior extremity. Forebody 246–891 (498) 333–454 (387) Paraschistorchis stenosoma and P. zancli are the most Forebody % 16.2–25.1 (20.2) 18.9–28.6 (23.9) similar species. Blend et al. (2017) invoked a distinc- Pre-oral L 18–63 (37) 19–41 (32) tion in the shape of the oral sucker (rounded to OS L 137–413 (291) 113–185 (145) elliptical vs funnel-shaped), for these two species, but OS W 146–406 (294) 137–179 (156) we have found the funnel-shape of P. zancli to be less OS L % 9.5–19.0 (12.1) 7.8–9.4 (8.6) distinctive in new specimens (reported below) than as Prepharynx L 17–41 (32) 9–15 (12) reported originally by Hanson (1953). Instead, we find Pharynx L 50–191 (112) 70–90 (79) a clear difference in the sucker ratios and the anterior Pharynx W 79–232 (156) 85–114 (109) extent of the vitelline follicles between the two VS L 108–321 (205) 136–169 (155) species. In addition to these morphological distinc- VS W 109–308 (207) 139–175 (159) tions, the hosts (only or dominant) of P. longivesicu- VS L / OS L 0.55–0.81 (0.71) 0.91–1.20 (1.11) lurus (Siganidae), P. seychellesiensis ? P. stenosoma VS W / OS W 0.57–0.81 (0.71) 0.98–1.03 (1.01) (Monacanthidae), and P. zancli (Zanclidae) appear to VS to ant. testis L 83–415 (247) 160–211 (182) be reliably diagnostic. Our newly generated molecular Testis no. 9–11 (10) 10–11 (11) data clearly distinguish P. seychellesiensis, P. steno- Ant. testis L 59–155 (111) 73–131 (101) soma and P. zancli. Ant. testis W 61–198 (114) 74–152 (121) Considering the distinctions outlined above, the Posttest. L 235–1312 (785) 299–351 (322) specimens reported from off Lizard and Heron Islands Posttest. L % 21.0–38.4 (30.5) 17.7–20.6 (19.2) are reliably identifiable as P. stenosoma. Notably, in Ovary L 53–213 (139) 74–120 (95) the cox1 dataset (Fig. 1), this species forms two sister Ovary W 58–196 (137) 70–107 (89) lineages, differing at 48 bp, each occurring off both Vitelline field L 858–3238 (1802) 435–592 (504) Heron and Lizard Islands; corresponding ITS2 and Previtelline L 390–962 (624) 544–611 (579) 28S sequences were identical (Figs. 2, 3). As discussed Postvitelline L 35–182 (72) 39–48 (44) above, we interpret all these specimens as representing Egg L 56–66 (62) 47–67 (55) P. stenosoma, but we have identified the genotype of Egg W 34–39 (36) 33–37 (35) lodged voucher hologenophores to enable future consideration of the issue. Our new records support the interpretation that C. pardalis, not S. fraenatum, is the main host for this Remarks species given that we have not detected it in 16 individuals of S. fraenatum, nor in five individuals of Paraschistorchis stenosoma, the type-species of S. bursa (Bloch & Schneider) or 153 S. chrysopterum, Paraschistorchis, was originally described as Sch. examined from Australian waters. The present record stenosoma from C. pardalis off Hawaii by Hanson is the first of this species from Australia. (1953). It was subsequently re-reported twice more from the same host/locality combination (Pritchard, Paraschistorchis seychellesiensis (Toman, 1989) Blend, Karar & Dronen, 2017 123 406 Syst Parasitol (2023) 100:381–413 Syn. Schistorchis seychellesiensis Toman, 1989 outline, dextral, typically slightly separated from Type-host: Cantherhines pardalis (Rüppell), Honey- ventral sucker anteriorly and anteriormost testis pos- comb filefish [reported as Cantherhines sandwichien- teriorly. Canalicular seminal receptacle saccular, sis (Quoy & Gaimard, 1824) Sandwich Isle file] antero-dorsal to ovary. Vitelline follicles extend from (Tetraodontiformes: Monacanthidae). about level of anterior margin of ventral sucker to Type-locality: Seychelles Islands, Indian Ocean. close to posterior extremity, anteriorly principally New material lateral to intestinal caeca, posteriorly occupying most Host: C. pardalis. available space around and posterior to testes, Localities: off Heron Island (2327’S, 15155’E), unevenly ventrally confluent posterior to testes, sep- southern GBR; off Lizard Island (1440’S, 14527’E), arated into relatively distinct columns by intestinal northern GBR, Australia. caeca, testes, and excretory vesicle. Uterus short, Site in host: Intestine. restricted to area lateral and anterior to ovary to ventral Prevalence: off Lizard Island 8 of 9 (89%). Off Heron sucker. Excretory vesicle I-shaped, reaches close to Island 14 of 35 (40%). level of posterior testis. Excretory pore terminal. Deposition of specimens: QM G240528–41. Representative DNA sequences: Partial cox1 mtDNA, Remarks seven sequences (two submitted to GenBank, OQ445545–46); ITS2 rDNA, seven sequences (two This species was described by Toman (1989) from C. submitted to GenBank, OQ442931–32); partial 28S pardalis from off the Seychelles Islands in the Indian rDNA, two sequences (one submitted to GenBank, Ocean based on just one adult and one immature OQ442914). specimen. The present material is from the same host, Measurements: Measurements in Table 7. broadly agrees with the original description of P. seychellesiensis, and is distinguished from the other Description (Fig. 7B) three species by the breadth of the body. Notably, the original description shows the testes as forming a [Based on 13 wholemount specimens, including three single column in contrast to the present specimens in hologenophores from C. pardalis from off Heron which the column commences as two testes wide and Island.] Body broad, linguiform, with maximum narrows to one for the last few testes. This distinction breadth at mid-hindbody; posterior margin sometimes translates into the post-testicular region in the original indented slightly at each anus; anterior margin description occupying a considerably smaller propor- rounded. Tegument spinous to mid-forebody. Pre-oral tion of total body length (16.3%) than in the present lobe well developed. Eye-spot pigment dispersed in specimens, 21.0–38.4 (30.5)%. Whether this differ- forebody. Oral sucker rounded in outline, muscular but ence should be considered as intra-specific or inter- with clear glandular components; U-shaped muscular specific is debateable. Here we take the view that the sphincter embedded at aperture, well-developed and similarities between these samples outweigh the prominent. Prepharynx short. Pharynx small, sub- differences and thus identify them as P. seychelle- quadrate, typically partly dorsal to oral sucker. siensis. Clearly, further sampling and, ideally, Oesophagus short but distinct in well-extended spec- sequencing from the type-location is necessary to imens. Intestinal bifurcation immediately anterior to improve confidence in this identification. ventral sucker. Caeca broad, terminate at separate ani Despite the seemingly obvious differences between at posterior extremity. Ventral sucker round in outline, P. stenosoma and P. seychellesiensis as indicated by unspecialised, noticeably smaller than oral sucker. the two figures presented here, the two are not always Testes 9–11, roughly ovoid, in broad contiguous as easily distinguished as might be expected. Inter- column, initially 2 testes wide but narrowing to one specific differences are most pronounced in the largest testis posteriorly; post-testicular region occupying specimens. In these, the two species differ clearly in approximately one-third body length. Seminal vesicle shape (P. seychellesiensis is much broader), oral saccular, small, postero-dorsal to ventral sucker. sucker width (in specimens of equivalent length, that Genital pore ventro-submedian, immediately anterior of P. seychellesiensis is significantly larger; the oral to ventral sucker, slightly sinistral. Ovary rounded in sucker of P. stenosoma never exceeds 251 lm in width 123 Syst Parasitol (2023) 100:381–413 407 in our samples but for P. seychellesiensis it ranges to Remarks 406 lm), the pharynx width tends to be larger in P. seychellesiensis, and the vitelline follicles commence Paraschistorchis zancli was originally described from further from the anterior end than do those of P. off Hawaii by Hanson (1953) from Z. cornutus based seychellesiensis. However, in specimens\2 mm long, on two specimens, and was redescribed based on a all these distinctions converge. We recognised just one larger sample collection from the same host/locality character distinction present in large easily distin- combination by Pritchard (1963). Yamaguti (1970) guished specimens (including the hologenophores) reported it from C. sandwichiensis (Monacanthidae) that was also present in smaller specimens. The partial off Hawaii. Lo et al. (2001) reported and figured it oral sucker sphincter of P. seychellesiensis is always from Z. cornutus from off Moorea (French Polynesia) discernible as a strongly and clearly recognisable and Heron Island (southern GBR) and reported structure whereas in P. stenosoma it is present, but far identical 28S and ITS2 sequences and only inconse- less easy to distinguish. Although the sphincters are quential differences in morphology. here figured as comparable, that of P. seychellesiensis The new specimens are consistent with the previous is consistently far better developed. morphological descriptions of the species with minor In molecular analysis, the present material consis- differences in appearance attributed to geographical or tently forms a single undivided clade for specimens intraspecific variation. A novel observation here is that from off Heron and Lizard Islands, in all cox1, ITS2 the supposedly key schistorchiine characteristic of a and 28S datasets (Fig. 1–3) and is clearly representa- partial sphincter embedded in the oral sucker is tive of a single species. This is the first record of this exceptionally hard to detect. We note that the first species from Australia. two descriptions of the species (Hanson, 1953; Pritchard, 1963) made no mention of any such Paraschistorchis zancli (Hanson, 1953) Blend, Karar structure. Yamaguti (1970) noted that ‘‘its gaping & Dronen, 2017 aperture is provided with a ring of fine muscle fibers’’ Syn. Schistorchis zancli Hanson, 1953 but gave no figure. Lo et al. (2001) did not describe the Type-host: Zanclus cornutus (Linnaeus), Moorish idol oral sucker. Careful examination of multiple speci- (Acanthuriformes, Zanclidae). mens from three localities (Heron Island, Palau, and Type-locality: Hawaii, USA. French Polynesia) leads us to conclude that a sphincter is probably present but that, unlike in its congeners, it New material is exceptionally difficult to discern. We further note Host: Z. cornutus. that, unlike in the congeners P. stenosoma and P. Localities: off Heron Island (2327’S, 15155’E), seychellesiensis, there is little if any development of southern GBR, Australia. Off Moorea (1729’S, gland cells in the oral sucker of this species. 14951’W), Society Archipelago, French Polynesia. Molecular data are identical for cox1 from French Off New Caledonia (2234’S, 16626’E). Off Palau Polynesia and Palau and for ITS2 from French (0720’N, 13429’E). Polynesia, Heron Island and Palau (Figs 1–2); no Site in host: Intestine. fresh samples were available for sequencing from the Prevalence: off Heron Island 3 of 30 (10%). Off GBR or New Caledonia. Moorea 9 of 15 (60%). Off New Caledonia 1 of 2 (50%). Off Palau 1 of 1 (100%). Deposition of specimens: QM G240542–71. Discussion Representative DNA sequences: Partial cox1 mtDNA, four sequences (two submitted to GenBank, Genera of Schistorchiinae OQ445547–48); ITS2 rDNA, two sequences (both submitted to GenBank, OQ442933–34); partial 28S As for most trematode taxa, generic diagnoses for the rDNA, one sequence (submitted to GenBank, Schistorchiinae are presently based exclusively on OQ442915). adult morphological characters. To distinguish six Measurements: Table 7. genera of Schistorchiinae, Blend et al. (2017) required variation in only three characters (oral sucker 123 408 Syst Parasitol (2023) 100:381–413 glandular or muscular; number of testes; and the would predict that the strongest gland cell develop- nature of the termination of intestinal caeca) to ment would be associated with strongly developed develop an effective system. However, because sphincters, and vice versa (as appears to be the case for trematodes have a limited number of characters, P. zancli). This idea suggests that some level of morphology may fail to clearly reflect phylogeny glandular development may be expected in almost all reliably (Bray & Cribb, 2012) and thus lead to a schistorchiines although it need not obviate its careful classification that is not natural. Here we recognise use in the distinction of genera. two genera in addition to those recognised by Blend et al. (2017), both strongly supported as phylogenet- Biogeography ically distinct but with relatively minor morphological distinction relative to the presently-recognised genera. Overall, what we know of the distribution of schis- Thus, it is concluded that the combination of two testes torchiine species fits a paradigm of broad Indo-west and intestinal caeca opening independently masks the Pacific distributions of trematodes of tropical marine phylogenetic distinction between species of Sphinc- fishes (e.g. Lo et al., 2001; Chambers & Cribb, 2006; teristomum and Lobatotrema and that the sharing of Cutmore et al., 2010; McNamara et al., 2012; Cutmore multiple testes and a highly glandular oral sucker & Cribb, 2022). This conclusion is weakened by the obscures that between species of Schistorchis and fact that there is little distributional evidence based on Blendiella. In our view, the combination of morpho- molecular data. However, the data presently available logical and molecular analyses results in a more (for L. aniferum and P. zancli) suggest that widespread nuanced and accurate classification, but not necessar- distributions are plausible. In addition, the species ily one that is easily applied under consideration are relatively morphologically One of the key characters used by Blend et al. distinctive. In this context there is evidence for the (2017) to distinguish schistorchiine genera, and still distribution of Sch. carneus in Sri Lanka, the Red Sea invoked by us, is the glandular vs muscular nature of and eastern Australia, for P. zancli from Hawaii, the oral sucker. Our new scheme recognises the French Polynesia, Palau and the GBR, for L. aniferum concepts of Blendiella, Lobatotrema, Schistorchis and from Fiji, Japan, New Caledonia, the GBR and Sphincteristomum as being partly defined by conspic- Ningaloo Reef, and for Sph. acollum from south-east uously glandular oral suckers and those of Paraschis- Asia and the GBR. Presumably we can expect more, torchis, Plesioschistorchis, Neomegacreadium perhaps most species to prove to be widespread as Machida & Kuramochi, 1999 and Sphincteristoma as collecting becomes more comprehensive. being partly defined by normally muscular oral Relative to the broad evidence for widespread suckers. We now suspect that this is an oversimplifi- species, there are several noteworthy exceptions. First, cation. Here we have observed clearly glandular the two new species of Blendiella have both been components in the oral suckers of two species of found only from off Lizard Island on the northern Paraschistorchis (P. stenosoma and P. seychellesien- GBR despite examination by us of a total of 145 sis), although not nearly as pronounced as those of the individuals of their three host species elsewhere, species of Blendiella, Lobatotrema, Schistorchis and including 87 from off Heron Island on the southern Sphincteristomum. In addition, we do not detect clear GBR. Evidently, not all the species occur everywhere gland cells at all in the oral sucker of P. zancli. that suitable hosts are found. Second, the two species Strikingly, the sphincter in the oral sucker of P. zancli known (mainly but not exclusively) from monacan- is reduced to the point that we have found it difficult to thids of the genus Cantherhines show enigmatic detect, although our view is that a weak structure is distributions. Paraschistorchis seychellesiensis was present. We hypothesise that glandular oral suckers described from the Seychelles and P. stenosoma was and the oral sphincter are linked characters of the described and subsequently reported several times Schistorchiinae as a whole. We suggest a functionality form Hawaii; neither species has been reported whereby the sphincter allows host tissue to be securely elsewhere. Here we found both species occurring in captured in the cavity of the oral sucker where the same individual fish on the GBR, more-or-less secretions of the gland cells begin some aspect of the halfway between the two original description sites. digestion of the host tissue. If broadly correct, we Molecular phylogenetic analysis finds that the two 123 Syst Parasitol (2023) 100:381–413 409 species are sister taxa. This relationship suggests the Pacific fish trematodes. The extent to which the possibility that the two species diverged in allopatry inconsistency of cox1 similarity is a reliable reflection (perhaps in cophyly with their definitive hosts, two of population history remains to be explored. species of Cantherhines although P. stenosoma also infects a species of Cantheschenia) and that their Acknowledgements We are grateful to many students and colleagues who facilitated this work and who have assisted in distributions subsequently expanded to now overlap. the collection of fishes and their trematodes over many years. Such hypotheses are clearly speculative and require We are highly appreciative of the support of the staff of Heron far more sampling, but the known patterns of distri- Island, Lizard Island, and Moreton Bay research stations. We are bution suggest that a range of processes may have especially grateful to Dr Toshiaki Kuramochi, Director of the Meguro Parasitological Museum, for providing photographs of affected the distributions of schistorchiines. many specimens in his care. This project represents a Finally, we draw attention to the varying patterns of contribution to Taxonomy Australia (2020), a national intraspecific structuring suggested by cox1 sequences. initiative organised under the auspices of the Australian This marker was sequenced for what we have inter- Academy of Science that brings together the taxonomic community to develop approaches that will significantly preted as five species over ranges at least as great as increase the rate at which new species are discovered, that between Heron and Lizard Islands which are resolved and named, with a view to completely documenting separated by about 1,200 km. Three patterns were the Australian biota within a generation. recognised. Paraschistorchis seychellesiensis and P. zancli showed negligible cox1 distinctions over range. Author contributions S.C. and T.C. collected the specimens. L.M., M.C. and T.C. did the morphological analysis. L.M. and In contrast, L. aniferum differed at 22 bp between S.C. did the molecular analyses. All authors contributed to samples from Lizard Island and Ningaloo Reef, from writing the manuscript. opposite sides of the Australian continent. Finally, S. carneus and P. stenosoma were each represented on Funding Open Access funding enabled and organized by the GBR by two cox1 populations, both differing at 48 CAUL and its Member Institutions. Collecting on the GBR was supported periodically by funding from the Australian Research bp; in the case of P. stenosoma, both populations occur Council and the Australian Biological Resources Study. at both Heron and Lizard Islands. The differences of Overall, this research was supported primarily by the 48 bp are only marginally smaller than the 56–57 bp Australian Biological Resources Study National Taxonomy between the two new species of Blendiella which are Research Grant RG19-37, awarded to SCC and THC. easily differentiated by testis number but are otherwise Data availability The data that support the findings of this highly similar. Contrasting patterns of regional pop- study are available from the corresponding author upon ulation structure and its absence have been reported reasonable request. for related species of Aporocotylidae (Cutmore et al., 2021), Bivesiculidae (Cribb et al., 2022), Lepocreadi- Declarations idae (Bray et al., 2018; 2022), and Monorchiidae Conflict of interest The authors declare that they have no (McNamara et al., 2014; Wee et al., 2022); we infer conflict of interest. that such discrepancies should now come as no surprise. In contrast, the finding of distinct, sympatric Ethical approval All applicable institutional, national, and international guidelines for the care and use of animals were cox1 lineages of the same species infecting the same followed. fish species remains uncommon; it was reported for two species of the lepocreadiid genus Preptetos by Open Access This article is licensed under a Creative Com- Bray et al. (2022). In that study, and here, the two cox1 mons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any med- populations are interpreted as representing the same ium or format, as long as you give appropriate credit to the species on the basis of the species recognition criteria original author(s) and the source, provide a link to the Creative proposed by Bray et al. (2022); they fail the test for Commons licence, and indicate if changes were made. The recognition as separate species because, although they images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated form reciprocally monophyletic lineages, at a deeper otherwise in a credit line to the material. If material is not level they are monophyletic, their hosts are the same, included in the article’s Creative Commons licence and your and they have indistinguishable morphology. Overall, intended use is not permitted by statutory regulation or exceeds we conclude that cox1 sequences frequently give the permitted use, you will need to obtain permission directly insight into the recent population history of Indo- 123 410 Syst Parasitol (2023) 100:381–413 from the copyright holder. To view a copy of this licence, visit Cribb, T. H., & Bray, R. A. (1999). A review of the Apocre- http://creativecommons.org/licenses/by/4.0/. adiidae Skrjabin, 1942 (Trematoda: Digenea) and description of Australian species. Systematic Parasitology, 44, 1–36. https://doi.org/10.1016/0005-2795(75)90348-7 Cribb, T. H., & Bray, R. A. (2010). Gut wash, body soak, References blender and heat-fixation: approaches to the effective col- lection, fixation and preservation of trematodes of fishes. Abid-Kachour, S., Mouffok, S., & Boutiba, Z. (2013). Systematic Parasitology, 76, 1–7. https://doi.org/10.1007/ Description of a new species of Sphincteristomum from s11230-010-9229-z sparid fishes of the Algerian coast (Western Mediter- Cribb, T. H., Bray, R. A., Justine, J.-L., Reimer, J., Sasal, P., ranean). Journal of Environmental Protection, 4, Shirakashi, S., & Cutmore, S. C. (2022). A world of tax- 1129–1136. https://doi.org/10.4236/jep.2013.410129 onomic pain: cryptic species, inexplicable host-specificity, Andres, M. J., Pulis, E. E., Curran, S. S., & Overstreet, R. M. and host-induced morphological variation among species (2018). On the systematics of some marine haploporids of Bivesicula Yamaguti, 1934 (Trematoda: Bivesiculidae) (Trematoda) with the description of a new species of from Indo-Pacific Holocentridae, Muraenidae and Ser- Megasolena Linton, 1910. Parasitology International, 67, ranidae. Parasitology, 149, 831–853. https://doi.org/10. 805–815. https://doi.org/10.1016/j.parint.2018.08.002 1017/s0031182022000282 Ankenbrand, M. J., Keller, A., Wolf, M., Schultz, J., & Forster, Curran, S. S., Tkach, V. V., & Overstreet, R. M. (2013a). F. (2015). ITS2 Database V: twice as much. Molecular Molecular evidence for two cryptic species of Homa- Biology and Evolution, 32, 3030–3032. https://doi.org/10. lometron (Digenea: Apocreadiidae) in freshwater fishes of 1093/molbev/msv174 the southeastern United States. Comparative Parasitology, Blend, C. K., Karar, Y. F. M., & Dronen, N. O. (2017). Revision 80, 186–195. https://doi.org/10.1654/4626.1 of the Megaperidae Manter, 1934 n. comb. (Syn. Apocre- Curran, S. S., Tkach, V. V., & Overstreet, R. M. (2013b). A new adiidae Skrjabin, 1942) including a reorganization of the species of Homalometron (Digenea: Apocreadiidae) from Schistorchiinae Yamaguti, 1942. Zootaxa, 4358, 1–44. fishes in the northern Gulf of Mexico. Journal of Para- https://doi.org/10.11646/zootaxa.4358.1.1 sitology, 99, 93–101. https://doi.org/10.1645/GE-3169.1 Bray, R. A., & Cribb, T. H. (2012). Reorganisation of the Cutmore, S. C., Bennett, M. B., & Cribb, T. H. (2010). Sta- superfamily Lepocreadioidea Odhner, 1905 based on an phylorchis cymatodes (Gorgoderidae Anaporrhutinae) inferred molecular phylogeny. Systematic Parasitology, from carcharhiniform, orectolobiform and myliobatiform 83, 169–177. https://doi.org/10.1007/s11230-012-9386-3 elasmobranchs of Australasia: Low host specificity, wide Bray, R. A., Cutmore, S. C., & Cribb, T. H. (2018). Lepotrema distribution and morphological plasticity. Parasitology Ozaki, 1932 (Lepocreadiidae: Digenea) from Indo-Pacific International, 59, 579–586. https://doi.org/10.1016/j. fishes, with the description of eight new species, charac- parint.2010.08.003 terised by morphometric and molecular features. System- Cutmore, S. C., & Cribb, T. H. (2022). New collections of blood atic Parasitology, 95, 693–741. https://doi.org/10.1007/ flukes (Aporocotylidae) from fishes of the tropical Indo- s11230-018-9821-1 west Pacific, including a new genus, two new species and Bray, R. A., Cutmore, S. C., & Cribb, T. H. (2022). A paradigm molecular evidence that Elaphrobates chaetodontis for the recognition of cryptic trematode species in tropical (Yamaguti, 1970) is widespread in the region. Parasitology Indo-west Pacific fishes: the problematic genus Preptetos International, 88, 102565, https://doi.org/10.1016/j.parint. (Trematoda: Lepocreadiidae). International Journal for 2022.102565 Parasitology, 52, 169–203. https://doi.org/10.1016/j. Cutmore, S. C., Yong, R. Q. Y., Reimer, J. D., Shirakashi, S., ijpara.2021.08.004 Nolan, M. J., & Cribb, T. H. (2021). Two new species of Bray, R. A., Waeschenbach, A., Cribb, T. H., Weedall, G. D., threadlike blood flukes (Aporocotylidae), with a molecular Dyal, P., & Littlewood, D. T. J. (2009). The phylogeny of revision of the genera Ankistromeces Nolan & Cribb, 2004 the Lepocreadioidea (Platyhelminthes, Digenea) inferred and Phthinomita Nolan & Cribb, 2006. Systematic Para- from nuclear and mitochondrial genes: Implications for sitology, 98, 641–664. https://doi.org/10.1007/s11230- their systematics and evolution. Acta Parasitologica, 54, 021-10002-8 310–329. https://doi.org/10.2478/s11686-009-0045-z Darriba, D., Taboada, G. L., Doallo, R., & Posada, D. (2012). Chambers, C. B., & Cribb, T. H. (2006). Phylogeny, evolution jModelTest 2: more models, new heuristics and parallel and biogeography of the Quadrifoliovariinae Yamaguti, computing. Nature Methods, 9, 772–772. 1965 (Digenea: Lecithasteridae). Systematic Parasitology, Edgar, R. C. (2004). MUSCLE: multiple sequence alignment 63, 61–82. https://doi.org/10.1007/s11230-005-9007-5 with high accuracy and high throughput. Nucleic Acids Cribb, T. H. (2005). Family Apocreadiidae Srjabin, 1942. In: A. Research, 32, 1792–1797. https://doi.org/10.1093/nar/ Jones, R. A. Bray and D. I. Gibson (Ed.), Keys to the gkh340 Trematoda Volume 2. Vol. 2. Wallingford: CAB Interna- Fayton, T. J., Curran, S. S., Andres, M. J., Overstreet, R. M., & tional and The Natural History Museum, pp. 621–640. McAllister, C. T. (2016). Two new species of Homa- Cribb, T. H., Anderson, G. R., Adlard, R. D., & Bray, R. A. lometron (Digenea: Apocreadiidae) from nearctic fresh- (1998). A DNA-based demonstration of a three-host life- water fundulids, elucidation of the life cycle of H. cycle for the Bivesiculidae (Platyhelminthes: Digenea). cupuloris, and molecular phylogenetic analysis of some International Journal for Parasitology, 28, 1791–1795. congeners. Journal of Parasitology, 102, 94–104. https:// https://doi.org/10.1016/S0020-7519(98)00127-1 doi.org/10.1645/15-862 123 Syst Parasitol (2023) 100:381–413 411 Hafeezullah, M. (1981). Schistorchiid trematodes of marine sequences. Molecular Phylogenetics and Evolution, 3, fishes of India, with considerations on the status of the 221–229. https://doi.org/10.1006/mpev.1994.1024 genus Megacreadium Nagaty, 1956 and family Schis- Littlewood, D. T. J., Curini-Galletti, M., & Herniou, E. A. torchiidae Yamaguti, 1942. Bulletin of the Zoological (2000). The interrelationships of Proseriata (Platy- Survey of India, 3, 167–177. helminthes: Seriata) tested with molecules and morphol- Hanson, M. L. (1953). A discussion of the trematode genus ogy. Molecular Phylogenetics and Evolution, 16, 449–466. Schistorchis (Family Lepocreadiidae) with descriptions of https://doi.org/10.1006/mpev.2000.0802 two new species from Hawaii. Pacific Science, 7, 447–452. Littlewood, D. T. J., Rohde, K., & Clough, K. A. (1997). Par- Hernandez-Mena, D. I., Cabanas-Granillo, J., Medina-Hernan- asite speciation within or between host species?—phylo- dez, E., & Perez-Ponce de Leon, G. (2022). Discovery of a genetic evidence from site-specific polystome new species of Homalometron Stafford, 1904 (Digenea: monogeneans. International Journal for Parasitology, 27, Apocreadiidae) from the stripped mojarra, Eugerres plu- 1289–1297. https://doi.org/10.1016/S0020- mieri in a coastal lagoon of the Gulf of Mexico. Journal of 7519(97)00086-6 Helminthology, 96, (e46), 1–8. https://doi.org/10.1017/ Lo, C. F., Morgan, J. A. T., Galzin, R., & Cribb, T. H. (2001). s0022149x22000372 Identical digeneans in coral reef fishes from French Poly- Hsieh, J.-F., Jeng, M.-L., Hsieh, C.-H., Ko, C.-C., & Yang, P.-S. nesia and the Great Barrier Reef (Australia) demonstrated (2014). Phylogenetic diversity and conservation of pro- by morphology and molecules. International Journal for tected click beetles (Campsosternus spp.) in Taiwan: a Parasitology, 31, 1573–1578. https://doi.org/10.1016/ molecular approach to clarifying species status. Journal of S0020-7519(01)00306-X Insect Conservation, 18, 1059–1071. https://doi.org/10. Lühe, M. (1906). Report on the trematode parasites from the 1007/s10841-014-9716-7 marine fishes of Ceylon. The Royal Society Report on the Huston, D. C., Cutmore, S. C., & Cribb, T. H. (2018). Isorchis Pearl Oyster Fisheries, 5, 97–108. cannoni n. sp. (Digenea: Atractotrematidae) from Great Machida, M., & Kuramochi, T. (1999). Digenean trematodes Barrier Reef rabbitfishes and the molecular elucidation of from tetraodontiform fishes from Japanese and adjacent its life cycle. J Helminthol, 92, 604–611. https://doi.org/10. waters. Bulletin of the National Science Museum, Tokyo, 1017/S0022149X17000906 Series A, Zoology, 25, 1–25. ICZN (2012). International Commission on Zoological Maddison, W. P., & Maddison, D. R. (2021). Mesquite: A Nomenclature: Amendment of articles 8, 9, 10, 21 and 78 modular system for evolutionary analysis, Version 3.31. of the International Code of Zoological Nomenclature to http://mesquiteproject.wikispaces.com/installation. expand and refine methods of publication. Bulletin of Madhavi, R., Narasimhulu, S. V., & Shameem, U. (1986). Zoological Nomenclature, 69, 161–169. Digenetic trematodes from marine fishes of Kalingapatnam Johnston, S. J. (1913). On some Queensland trematodes, with coast, Bay of Bengal. Families Lepocreadiidae, Deropris- anatomical observations and descriptions of new species tiidae and Schistorchiidae. Rivista di Parassitologia, 47, and genera. Quarterly Journal of Microscopical Science, 111–119. 59, 361–400. Maggioi, D., Arrigoni, R., Galli, P., Berumen, M. L., Seveso, D., Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., & Montano, S. (2018). Polyphyly of the genus Zanclea and Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, family Zancleidae (Hydrozoa, Capitata) revealed by the C., Thierer, T., Ashton, B., Meintjes, P., & Drummond, A. integrative analysis of two bryozoan-associated species. (2012). Geneious Basic: an integrated and extendable Contributions to Zoology, 87, 87–104. https://doi.org/10. desktop software platform for the organization and analysis 1163/18759866-08702003 of sequence data. Bioinformatics, 28, 1647–1649. https:// Manter, H. W. (1963). Studies on digenetic trematodes of fishes doi.org/10.1093/bioinformatics/bts199 of Fiji. II. Families Lepocreadiidae, Opistholebetidae, and Keller, A., Schleicher, T., Schultz, J., Muller, T., Dandekar, T., Opecoelidae. Journal of Parasitology, 49, 99–113. & Wolf, M. (2009). 5.8S-28S rRNA interaction and HMM- McCord, C. L., & Westneat, M. W. (2016). Phylogenetic rela- based ITS2 annotation. Gene, 430, 50–57. https://doi.org/ tionships and the evolution of BMP4 in triggerfishes and 10.1016/j.gene.2008 filefishes (Balistoidea). Molecular Phylogenetics and Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: Evolution, 94, 397–409. https://doi.org/10.1016/j.ympev. Molecular Evolutionary Genetics Analysis version 7.0 for 2015.09.014 bigger datasets. Molecular Biology and Evolution, 33, McNamara, M. K. A., Adlard, R. D., Bray, R. A., Sasal, P., & 1870–1874. https://doi.org/10.1093/molbev/msw054 Cribb, T. H. (2012). Monorchiids (Platyhelminthes: Kurochkin, Y. V. (1974). On new species of the trematode genus Digenea) of chaetodontid fishes (Perciformes): biogeo- Schistorchis (Trematoda, Schistorchidae) from monacan- graphical patterns in the tropical Indo-West Pacific. Par- thids of the Great Australian Bight. Proceedings of Pacific asitology International, 61, 288–306. https://doi.org/10. Ocean Scientific Research Institute for Fisheries Man- 1111/j.1432-1033.1975.tb02184.x agement and Oceanography, 88, 67–68. McNamara, M. K. A., Miller, T. L., & Cribb, T. H. (2014). Lehr, E., Moravec, J., & von May, R. (2020). A new cryptic Evidence for extensive cryptic speciation in trematodes of genus of terrestrial lizard (Gymnophthalmidae: Cer- butterflyfishes (Chaetodontidae) of the tropical Indo-West cosaurinae) from the eastern Andes of central Peru. Sala- Pacific. International Journal for Parasitology, 44, 37–48. mandra, 56, 1–15. https://doi.org/10.1002/j.1879-3479.1975.tb00331.x Littlewood, D. T. J. (1994). Molecular phylogenetics of cupped Miller, M. A., Pfeiffer, E., & Schwartz, T. (2010). Creating the oysters based on partial 28S ribosomal RNA gene CIPRES Science Gateway for inference of large 123 412 Syst Parasitol (2023) 100:381–413 phylogenetic trees. In: Proceedings of the Gateway Com- Pietrasiak, N., Reeve, S., Osorio-Santos, K., Lipson, D. A., & puting Environments Workshop (GCE), 14 Nov. 2010, Johansen, J. R. (2021). Trichotorquatus gen. nov. - a new New Orleans, LA, pp. 1–8. https://doi.org/10.1109/GCE. genus of soil cyanobacteria discovered from American 2010.5676129 drylands. Journal of Phycology, 57, 886–902. https://doi. Miller, T. L., & Cribb, T. H. (2013). Dramatic phenotypic org/10.1111/jpy.13147 plasticity within species of Siphomutabilus n. g. (Digenea: Pleijel, F., Jondelius, U., Norlinder, E., Nygren, A., Oxelman, Cryptogonimidae) from Indo-Pacific caesionines (Perci- B., Schander, C., Sundberg, P., & Thollesson, M. (2008). formes: Lutjanidae). Systematic Parasitology, 86, Phylogenies without roots? A plea for the use of vouchers 101–112. https://doi.org/10.1007/s11230-013-9436-5 in molecular phylogenetic studies. Molecular Phyloge- Morgan, J. A. T., & Blair, D. (1995). Nuclear rDNA ITS netics and Evolution, 48, 369–371. https://doi.org/10.1016/ sequence variation in the trematode genus Echinostoma: an j.ympev.2008.03.024 aid to establishing relationships within the 37-collar-spine Pritchard, M. H. (1963). Studies on digenetic trematodes of group. Parasitology, 111, 609–615. https://doi.org/10. Hawaiian fishes, primarily families Lepocreadiidae and 1017/S003118200007709X Zoogonidae. Journal of Parasitology, 49, 578–587. Myers, R. F. (1991). Micronesian reef fishes. A practical guide Pulis, E. E., Curran, S. S., Andres, M. J., & Overstreet, R. M. to the identification of the coral reef fishes of the tropical (2014). Change in rank of Megaperidae (Trematoda) to central and western Pacific. Barrigada: Coral Graphics. Megaperinae within the Apocreadiidae and description of Odhner, T. (1928). Veitere Trematoden mit Anus. Arkiv für Haintestinum amplum n. g., n. sp. Parasitology Interna- Zoologi, 20, 1–6. tional, 63, 269–274. https://doi.org/10.1016/j.parint.2013. Olson, P. D., Cribb, T. H., Tkach, V. V., Bray, R. A., & Lit- 11.007 tlewood, D. T. J. (2003). Phylogeny and classification of Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., the Digenea (Platyhelminthes: Trematoda). International Darling, A., Hohna, S., Larget, B., Liu, L., Suchard, M. A., Journal for Parasitology, 33, 733–755. https://doi.org/10. & Huelsenbeck, J. P. (2012). MrBayes 3.2: Efficient 1016/S0020-7519(03)00049-3 Bayesian phylogenetic inference and model choice across a Oshmarin, P. G., Mamaev, Y. L., & Parukhin, A. M. (1961). large model space. Systematic Biology, 61, 539–542. New species of trematodes of the family Diploprocto- https://doi.org/10.1093/sysbio/sys029 daeidae Ozaki, 1928. Helminthologia, 3, 254–261. Shalygin, S., Shalygina, R., Johansen, J. R., Pietrasiak, N., Pantoja, C., Scholz, T., Luque, J. L., & Deleon, G. P. P. (2021). Berrendero Gomez, E., Bohunicka, M., Mares, J., & Sheil, Molecular and morphological evidence of a new species of C. A. (2017). Cyanomargarita gen. nov (Nostocales, Crassicutis Manter 1936 (Digenea), a parasite of cichlids in Cyanobacteria): convergent evolution resulting in a cryptic South America. Parasitology Research, 120, 2429–2443. genus. Journal of Phycology, 53, 762–777. https://doi.org/ https://doi.org/10.1007/s00436-021-07161-4 10.1111/jpy.12542 Parker, J. H., Curran, S. S., Overstreet, R. M., & Tkach, V. V. Snyder, S. D., & Tkach, V. V. (2001). Phylogenetic and bio- (2010). Examination of Homalometron elongatum Manter, geographical relationships among some Holarctic frog lung 1947 and description of a new congener from Eucinosto- flukes (Digenea: Haematoloechidae). Journal of Para- mus currani Zahuranec, 1980 in the Pacific Ocean off sitology, 87, 1433–1440. https://doi.org/10.1645/0022- Costa Rica. Comparative Parasitology, 77, 154–163. 3395(2001)087[1433:PABRAS]2.0.CO;2. https://doi.org/10.1654/4451.1 Stamatakis, A. (2014). RAxML version 8: a tool for phyloge- Parukhin, A. M. (1963). [New species of trematodes from fishes netic analysis and post-analysis of large phylogenies. of south China Sea] (In Russian). Moskova, 17, 123–125. Bioinformatics, 30, 1312–1313. https://doi.org/10.1093/ Parukhin, A. M. (1970). [New species of trematodes in fish from bioinformatics/btu033 the Red Sea] (In Russian). Biologiya Morya, Kiev, 20, Swofford, D. L. (2002). PAUP*. Phylogenetic Analysis using 267–272. Parsimony (*and other methods) version 4. Sunderland, Parukhin, A. M. (1989). [Parasitic worms of bottom fishes of the Massachusetts, Sinauer Associates. southern seas]. (In Russian) Kiev: Naukova Dumka. Toman, G. (1989). Digenetic trematodes of marine teleost fishes Parukhin, A. M., & Chikunova, D. A. (1964). Helminth fauna of from the Seychelles, Indian Ocean. II. Acta Parasitologica marine fish of the family Balistidae from the South China Polonica, 34, 235–246. Sea. Rivista di Parassitologia, 36, 100–115. Wee, N. Q.-X., Cribb, T. H., Bray, R. A., & Cutmore, S. C. Pérez-Ponce de León, G., & Hernandez-Mena, D. I. (2019). (2017). Two known and one new species of Proctoeces Testing the higher-level phylogenetic classification of from Australian teleosts: variable host-specificity for clo- Digenea (Platyhelminthes, Trematoda) based on nuclear sely related species identified through multi-locus molec- rDNA sequences before entering the age of the ‘‘next- ular data. Parasitology International, 66, 16–26. https:// generation’ Tree of Life. Journal of Helminthology, 93, doi.org/10.1016/j.parint.2016.11.008 260–276. https://doi.org/10.1017/s0022149x19000191 Wee, N. Q. X., Cribb, T. H., Shirakashi, S., & Cutmore, S. C. Pérez-Ponce de León, G., Razo-Mendivil, U., & Garcia- (2022). Three new species of Helicometroides Yamaguti, Magana, L. (2012). Morphological and molecular evi- 1934 from Japan and Australia, with new molecular evi- dences for the existence of two new species of Homa- dence of a widespread species. Parasitology, 149, lometron (Digenea: Apocreadiidae), parasites of cichlids 622–639. https://doi.org/10.1017/S0031182022000051 (Osteichthyes: Cichlidae). Zootaxa, 37–48. https://doi.org/ Xia, X. (2018). DAMBE7: new and improved tools for data 10.11646/ZOOTAXA.3407.1.3 analysis in molecular biology and evolution. Molecular 123 Syst Parasitol (2023) 100:381–413 413 Biology and Evolution, 35, 1550–1552. https://doi.org/10. Yamaguti, S. (1971). Synopsis of Digenetic Trematodes of 1093/molbev/msy073 Vertebrates. Tokyo: Keigaku Publishing Co. Xia, X., & Lemey, P. (2009). Assessing substitution saturation Yong, R. Q. Y., Cribb, T. H., & Cutmore, S. C. (2021). with DAMBE. In: P. Lemey, M. Salemi and A. M. Van- Molecular phylogenetic analysis of the problematic genus damme (Ed.), Phylogenetic Handbook: A Practical Cardicola (Digenea: Aporocotylidae) indicates massive Approach to Phylogenetic Analysis and Hypothesis Test- polyphyly, dramatic morphological radiation and host- ing, 2nd Edition. Vol. Cambridge: Cambridge University switching. Molecular Phylogenetics and Evolution, 164, Press, pp. 615–630. 107290. https://doi.org/10.1016/j.ympev.2021.107290 Xia, X., Xie, Z., Salemi, M., Chen, L., & Wang, Y. (2003). An index of substitution saturation and its application. Publisher’s Note Springer Nature remains neutral with Molecular Phylogenetics and Evolution, 26, 1–7. https:// regard to jurisdictional claims in published maps and doi.org/10.1016/s1055-7903(02)00326-3 institutional affiliations. Yamaguti, S. (1970). Digenetic Trematodes of Hawaiian Fishes. Tokyo: Keigaku Publishing Co. 123