Proposal of Troglocephalinae n. subfam. (Monogenea: Monocotylidae) to accommodate existing and two new monocotylids from the gills of rhinopristiform shovelnose rays

Troglocephalinae n. subfam. is proposed for Spinuris Doran, 1953, Neoheterocotyle Hargis, 1955, Anoplocotyloides Young, 1967, Troglocephalus rhinobatidis Young, 1967 (previously incertae sedis), Nonacotyle pristis Ogawa, 1991, Mehracotyle insolita Neifar, Euzet & Ben Hassine, 2002, Scuticotyle cairae n. gen. et sp., and Brancheocotyle imbricata n. gen. et sp. All members of the proposed new subfamily are gill parasites of shovelnose rays of the order Rhinopristiformes. The subfamilies Heterocotylinae Chisholm, Wheeler & Beverley-Burton, 1995, and Dasybatotreminae Bychowsky, 1957, are amended to exclude Spinuris, Nonacotyle, Neoheterocotyle, and Anoplocotyloides and Mehracotyle, respectively. Heterocotylinae includes gill parasites of members of the orders Myliobatiformes and Torpediniformes. Dasybatotreminae includes parasites of the gills and pharyngeal cavity of members of the orders Myliobatiformes and Rajiformes. A revised phylogeny of the Monocotylidae Taschenberg, 1879 is presented and discussed, based on 28S rDNA sequences, including new sequences for Myliocotyle pteromylaei Neifer, Euzet & Ben Hassine, 1999, Heterocotyle tokoloshei Vaughan & Chisholm, 2010, Neoheterocotyle robii Vaughan & Chisholm, 2010, and the two newly proposed species and genera. Additional locality records are also provided for Monocotylidae from off South Africa. Supplementary Information The online version contains supplementary material available at 10.1007/s11230-024-10174-z.


Introduction
Monocotylids (Monogenea: Monocotylidae Taschenberg, 1879) are parasites of chondrichthyans of marine, brackish and fresh waters.Their host microhabitats are diverse, including the gill lamellae, pharyngeal cavity, skin surface, nasal tissue, urogenital system, and inner wall of the body cavity (Chisholm and Whittington 1998a;Derouiche et al. 2019, Bullard et al. 2021;Ruiz-Escobar et al. 2022).Traditionally, the morphology of the haptor, including the number of loculi (or a 3-part attachment organ; Bullard et al. 2021), and the presence of a variety of ventral and dorsal haptoral structures, presumably to facilitate attachment to the variety of host microhabitats, has been of primary importance in discriminating higher-level monocotylid taxa (Chisholm and Whittington 1998a;Bullard et al. 2021).The morphology of the male copulatory organ and vagina is useful for discriminating between species.Comparatively little attention has been afforded to the importance of the anterior head region, and its structures that might demonstrate relatedness between taxa.An inconsistent approach exists for including details of the head glands of the anterior head region historically.Some additional details, such as the presence of ventral pits in the anterior head region (e.g., Hargis 1955;Young 1967, for Neoheterocotyle Hargis, 1955 species) or differences in the nature of the gland-duct openings, have largely been ignored.Notably, Young (1967) described both Neoheterocotyle rhinobatidis (Young, 1967) Chisholm, 1994 andTroglocephalus rhinobatidis Young, 1967 in the same work, yet only described ventral pits for the latter.This feature is confirmed in all Neoheterocotyle species (Chisholm and Whittington 1997).Young (1967) also referred to the ventral pits as "clear markings of unknown nature" for Anoplocotyloides papillatus (Doran, 1953), Young, 1967, perhaps because the function of these ventral pits has never adequately been experimentally demonstrated.Some of these subtle characters are undoubtedly difficult to observe, but with current, modern technology, a renewed focus on this region in monocotylids is warranted.Recently, workers have begun to include the relative importance of these subtle characters in phylogenetic analyses of the family (e.g., Boeger et al. 2014;Bullard et al. 2021).
Dasybatotreminae was originally erected by Bychowsky (1957) to accommodate Dasybatotrema dasybatis (MacCallum, 1916), Price, 1938 from the gills of the marine rays Dasyatis pastinaca (Linnaeus) and Pastinachus centrourus (Mitchill) [now Bathytoshia centroura (Mitchill)].The proposal of Dasybatotreminae was based on the presence of numerous anterior gland-duct openings along the anterior edge of the "adoral sucker" (anterior head region), and the morphology of the hamulus consisting of a reduced superficial root and elongated deep root in the typespecies.Yamaguti (1963) subsequently assigned D. dasybatis to Monocotylinae, thus synonymising Dasybatotreminae with the former subfamily, without explanatory comment.Chisholm et al. (1995) revised the family based on morphology, reinstated Dasybatotreminae, and included the additional genera Anoplocotyloides, Timofeevia, and Troglocephalus based on two apomorphies: the elongated deep root of the hamulus, and numerous anterior gland-duct openings.The inclusion of Tr. rhinobatidis in Dasybatotreminae would later prove problematic.In the subsequent molecular phylogenetic analysis of the family by Chisholm et al. (2001a), as the sole representative of Dasybatotreminae, Tr. rhinobatidis grouped together with three Neoheterocotyle species in a monophyletic group, separate to Heterocotyle capricornensis Chisholm & Whittington, 1996.This presented the Heterocotylinae as paraphyletic but also suggested that the presence of ventral pits on the anterior head region of Tr. rhinobatidis and Neoheterocotyle species was a unifying character.Given the low taxa resolution of this initial molecular phylogeny, the monotypic nature of Troglocephalus, and to avoid transferring Tr. rhinobatidis to Heterocotylinae, Tr. rhinobatidis was rendered incertae sedis (Chisholm et al. 2001a).A year later, another Troglocephaluslike monocotylid, Mehracotyle insolita Neifar, Euzet & Ben Hassine, 2002 was proposed by Neifar et al. (2002).These authors were aware of the problems resulting from profound changes to the classification of Heterocotylinae if Tr. rhinobatidis was transferred to this subfamily, and specifically to avoid these problems, they chose to consider Me. insolita a member of Dasybatotreminae based on the earlier morphological work by Chisholm et al. (1995).In making their decision regarding Me. insolita, they stated that it was provisional, and stopped short of formally reassigning Troglocephalus back to Dasybatotreminae.In the same work, Neifar et al. (2002) also indicated that the presence of ventral pits on the anterior head region in Me. insolita and Tr.rhinobatidis were also shared with A. papillatus, and that all three species were from rhinopristiform hosts.In addition, these authors stated that the larvae observed for Me.insolita demonstrated a morphological similarity in the permutation of larval ciliated cells with larvae described for Neoheterocotyle species, a similar conclusion reached by Chisholm (1998) for larvae of Ne. rhinobatidis and Tr.rhinobatidis, suggesting a close relationship.This close relationship was also supported by the investigations of spermiogenesis and sperm ultrastructure by Watson (1997) for the latter two species.
Both Dasybatotrema species, D. dasybatis and D. spinosum Timofeeva, 1983 have a distinct arrangement of gland-duct openings of the anterior head region to those of other monocotylids and have no ventral pits.The unique morphology of these glandduct openings is also shared by Timofeevia rajae (Timofeeva, 1983) Chisholm, Wheeler & Beverley-Burton, 1995, which also has no ventral pits.Recently, Dasybatotreminae was amended to accommodate a new genus, Peruanocotyle Chero, Cruces, Sáez & Luque, 2018, for P. chisholmae Chero, Cruces, Sáez & Luque, 2018.A second species of Peruanocotyle, P. pelagica Ruiz-Escobar, Torres-Carrera & Ramos-Sánchez, 2022 was subsequently described.Both Peruanocotyle species demonstrate the same anterior head region gland-duct opening arrangement as Dasybatotrema species and Ti.rajae, and the absence of ventral pits.Except for Ti.rajae as a parasite of a rajiform skate, these species are all parasites of myliobatiform stingrays.Dasybatotreminae can thus be separated into two groups of taxa based on the morphology of the anterior head region.Similarly, additional taxa from rhinopristiform hosts that share morphological similarities in the anterior head region with A. papillatus, Me. insolita and Tr.rhinobatidis, are currently classified under Heterocotylinae.Of these, only Neoheterocotyle species are represented currently in molecular phylogenies, but consistently reflect the original representation of Heterocotylinae as polyphyletic, remaining separate from other heterocotylinid species currently, even as family and subfamily resolution has improved over time (see Fehlauer-Ale and Littlewood 2011;Boeger et al. 2014;Vaughan et al. 2016;Derouiche et al. 2019;Bullard et al. 2021;Chero et al. 2021;Dalrymple et al. 2022;Ruiz-Escobar et al. 2022).
During the parasitological investigation of elasmobranchs off South Africa, two new monocotylid species representing new genera were collected from the shovelnose ray, Acroteriobatus annulatus (Smith).These monocotylids share morphological similarities of the anterior head region with A. papillatus, Me. insolita, Neoheterocotyle species, No. pristis, Spinuris species, and Tr.rhinobatidis.Based on the shared morphological features of the anterior head region in these species from rhinopristiform hosts, additional historic evidence in the literature of larval similarity between Tr. rhinobatidis, Neoheterocotyle species, and Me.insolita, similar sperm ultrastructure for Ne.rhinobatidis and Tr.rhinobatidis, and historic and new molecular phylogenetic information, a new subfamily is proposed herein, requiring the re-evaluation of Heterocotylinae and Dasybatotreminae.The two new species are described, and additional data are presented for other monocotylids collected during the same period, from South Africa.

Materials and methods
In April 2008, during the South African government department of Fisheries and Environment's commercial demersal sole fishery survey, two female and one male Dasyatis chrysonota (Smith), and one male Galeorhinus galeus (Linnaeus) were collected as dead trawl bycatch on board the fisheries research vessel Africana off South Africa's South coast (off Cape Agulhas).The gill arches were dissected, and nasal fossae inspected on board the Africana.Specimens of Heterocotyle pastinacae Scott, 1904 were recovered from the gills of D. chrysonota, and Cathariotrema selachii (MacCallum, 1916) from the nasal fossae of G. galeus.In February 2010, six Ac.annulatus, and a single, large female Aetomylaeus bovinus (Geoffroy Saint-Hilaire) were obtained live from the traditional commercial seine-net fishermen operating off Muizenberg beach (34°06'13" S, 18°29'00" E) in False Bay, South Africa, collected by staff of Two Oceans Aquarium, Cape Town.The Ae. bovinus and one Ac.annulatus succumbed to netting damage sustained during initial capture and died during transportation back to the aquarium and were subsequently dissected for parasites upon arrival.The remaining Ac. annulatus rays were housed in the aquarium's quarantine facility.After 24 hours, sampling of detritus from the bottom of the quarantine tank revealed the presence of monogenean eggs.Three rays were randomly removed for non-invasive parasitological examination using the method of Vaughan and Chisholm (2010a).Each of the three rays selected for the non-invasive treatment method was removed to a separate glass tank of 100 L volume, anaesthetised with 0.15 ml/L 2-phenoxyethanol, weighed, and given the anthelmintic praziquantel at 150 mg/kg by gavage (Vaughan and Chisholm 2010a).Thereafter, the volume of each 100 L tank was filtered through a 23 µm sieve to recover any monogeneans.The filtrate from each tank was placed into a separate glass inspection bowl with fresh, filtered seawater and observed under an Olympus SZ60 stereo zoom dissection microscope.Anaesthetised rays made a full recovery in separate holding tanks of fresh, filtered seawater.Live specimens of two unidentified species of Monocotylidae that originated from the gills, were recovered from the three treated, and one dissected Ac. annulatus rays; live specimens of two known representatives of Heterocotylinae were recovered from the gills of Ae.Bovinus: Myliocotyle pteromylaei Neifer, Euzet & Ben Hassine, 1999, and Heliocotyle kartasi Neifar, Euzet & Ben Hassine, 1999.Monogeneans that were recovered from between the gill lamellae of the dissected Ac. annulatus ray, were initially observed alive in a glass inspection bowl containing fresh filtered seawater or individually on microscope slides in a drop of fresh, filtered seawater.Observations were recorded and photomicrographs taken of the live monogeneans.Thereafter, monogeneans were individually preserved flat in analytical reagent grade absolute ethanol (ARE).Some monogeneans were preserved unflattened in ARE for DNA extraction and for full or partial proteolytic digestion of haptoral armature and reproductive structures, following the methodology of Vaughan et al. (2008).Those for proteolytic digestion were rehydrated with freshwater and placed individually onto glass microscope slides.Their haptor was severed from the body-proper and treated separately to the body on the same slide before being mounted in glycerine jelly under a coverslip sealed with clear nail varnish.Flat-preserved monogeneans for staining were rehydrated in freshwater before being stained with either Alum Carmine or diluted Gormori's Trichrome solutions, dehydrated in a graded ethanol series, cleared in Cedarwood oil, and permanently mounted individually in Canada balsam on glass microscope slides beneath a glass coverslip.Live monogeneans in temporary mounts of seawater, permanently mounted monogeneans and partial or total digests were examined using an Olympus CX 41 or Nikon Eclipse 200 compound light microscopes fitted with phase-contrast and dark-field optics.Photomicrographs were taken with an Olympus Altra 20 digital microscope camera mounted to the Olympus CX41 and drawings were made with the aid of a drawing tube.All measurements were taken using Olympus AnalySIS5 software calibrated to the Altra 20 and CX41.These measurements are given in micrometres as the mean ± standard deviation, followed in parenthesis by the range and the number of specimens measured.
DNA was extracted from two individual specimens of the two new species and from individuals of Myliocotyle pteromyleai Neifer, Euzet & Ben Hassine, 1999 (n = 1), Heterocotyle tokoloshei Vaughan & Chisholm, 2010 (n = 2), Electrocotyle whittingtoni Vaughan, Chisholm & Hansen, 2016 (n = 2) and Neoheterocotyle robii Vaughan & Chisholm, 2010 (n = 2) using the Mole DNA tissue kit on a MoleGenetics DNA extraction robot.PCR reactions were done using Illustra PuReTaq Ready-To-Go™ PCR Beads (GE Healthcare) in accordance with the manufacturer's instructions.Each reaction contained 1 μl of the forward primer, 1 μl of the reverse primer, 3μl of the template DNA and 20 μl of sterile water.The primer pair (C1 forward ACC CGC TGA ATT TAA GCA T; D2 reverse TGG TCC GTG TTT CAA GAC ) (Hassouna et al. 1984, see Chisholm et al. 2001a, b), was used to amplify a ∼950-bp fragment of the large subunit (LSU/28S) ribosomal DNA.A shorter fragment was also amplified using the primer combination Rob1 (Chisholm et al. 2001a) /D2.The PCR protocol was as follows: 4 min at 95 °C followed by 35 cycles of 1 min at 95°C, 1 min at 55°C, and 2 min at 72°C.PCR products were shipped to Macrogen Inc. (Seoul, Korea) for purification and sequencing on an Applied Biosystems 3730xl DNA Analyzer.All reactions were sequenced using the PCR primers.Sequence assembly and analysis of chromatograms were performed with Geneious Prime® version 2023.0.1 (www.genei ous.com).All nucleotide sequence data were deposited in GenBank under accession numbers KT735368-KT735369 and OR351731-OR351739 (see Table 1).To reveal possible identity with other species present in GenBank, all obtained sequences were submitted to a BlastN search (Zhang et al. 2000) using default parameter settings.
The phylogenetic analyses included the new sequences obtained in the present study in addition to all sequences taken from nominal species of Monocotylidae that were available (Table 1).Sequences of Capsala martinieri Bosc, 1811 (accession number AF382053), Entobdella hippoglossi (Müller, 1776) (accession number AY486151) Blainville, 1818, and Benedenia lutjani Whittington & Kearn, 1993 (accession number AY033939), were used as the outgroup, which is the same used by Bullard et al. (2021).For species for which several identical sequences were available, only the longest of these identical sequences was included.In addition, some sequences retrieved from GenBank were evaluated to be too short to be included (e.g., Triloculotrema sp.AF387512, see Bullard et al., 2021).The final selection consisted of 79 unique sequences representing 62 species, including the outgroup.Alignment of the selected SSU sequences was constructed using MAFFT v7 online (L-INS-I algorithm) (Katoh et al. 2019), resulting in a final alignment of 1236 nucleotide sites (Supplementary file S1).Maximum-likelihood (ML) phylogenetic trees of the alignment were constructed with W-IG-TREE (Trifinopoulos et al. 2016) accessible from http:// iqtree.cibiv.univie.ac.at.W-IG-TREE automatically determined the best-fit substitution models to be GTR+F+I+G4 according to the Bayesian information criterion.Branch support was assessed by ultrafast bootstrap (UFBoot2; Hoang et al. 2018) with the number of bootstrap alignments and the maximum number of iterations set to 10000.For Ultrafast bootstrap support, we consider values of 95% or higher as statistically significant and indicative of a well-supported group, while those with lower values are not considered significant.Finally, the sequence alignment was also processed with MEGAX (Stecher et al. 2020) to calculate the number of pairwise base differences (Supplementary file S2).
Specimens of C. selachii, Het.pastinacae, Hel.kartasi and My.pteromylaei were identified using Bullard et al. (2021), Neifar et al. (1998;1999a, b) or Chisholm (1995).The type series and vouchers of both new taxa described herein, the vouchers of C. selachii, and three vouchers of Het.pastinacae are deposited in the Australian Helminthological Collection (AHC) at the South Australian Museum in Adelaide, South Australia, Australia.Vouchers of Hel.kartasi and My.pteromylaei, and one voucher of Het.pastinacae are deposited in the Iziko South African Museum, Cape Town, South Africa (SAMC).The following museum specimens were examined for comparative purposes: British Museum of Natural History, Mehracotyle insolita (BMNH 2001.8.6

Remarks
The Heterocotylinae was originally proposed by Chisholm et al. (1995) to accommodate all monocotylids with one central and seven, eight or nine peripheral haptoral loculi that possess at least four sclerotised (or partially sclerotised) dorsal haptoral structures on the posterior and posterolateral loculi.Subsequently, the genera Heliocotyle and Malalophus were included, with members that have a single, rounded, partially sclerotised dorsal haptoral accessory structure on the posterior loculus (Neifar et al. 1999a;Chisholm and Whittington 2009) 2021) recently proposed three morphological groups for the Heterocotylinae genera.Group 1 included Electrocotyle, Heterocotyle, Myliocotyle, Potamotrygonocotyle, and Spinuris.This group consisting of those representatives with one central and eight peripheral loculi -two of which are interhamular, four "flaps" (= dorsal haptoral accessory structures) dorsal to the four posterior-most loculi, the absence of ventral locular-surface ridges, and the presence of a septal ridge in all but Spinuris.Group 2 included Neoheterocotyle, Septisinus [sic; = Septesinus], and Nonacotyle, with one central and seven peripheral loculi -one interhamular, three "flaps" dorsal to the three posterior-most loculi, the absence of ventral locular-surface ridges and septal ridges.Group 3 included Malalopholus [sic; = Malalophus] and Heliocotyle, with one central and seven peripheral loculi, one "flap" dorsal to the posterior loculus, the presence of ventral locular-surface ridges, and the absence of a septal ridge.Unfortunately, these groups are inaccurate.The first group excluded Po. quadracotyle Domingues, Pancera & Marques, 2007 with one central and four peripheral loculi, where a pair of dorsal haptoral accessory structures are present on the single interhamular posterior loculus, and a single bilobed dorsal haptoral accessory structure is present on each of the two lateral loculi (Domingues et al. 2007).Group 1 also excluded Po. septemcotyle Domingues & Marques, 2011, which has seven peripheral loculi (Domingues and Marques 2011).Nonacotyle in the second group has nine peripheral loculi, not seven, and has six dorsal haptoral accessory sclerites, two dorsal to the posterior loculus, and two dorsal to the two posterolateral loculi; Neoheterocotyle species have four or six dorsal haptoral accessory sclerites, two dorsal to the posterior loculus, and one or two dorsal to the two posterolateral loculi.Septesinus has four dorsal haptoral accessory structures, two on the posterior loculus, and one on both posterolateral loculi.All genera in groups 2 and 3 have a present septal ridge.
The monophyly of the Heterocotylinae, excluding Het.capricornensis, is well supported in our phylogeny (Fig. 1), which includes new sequences for Het.tokoloshei and My.pteromylaei.Heterocotyle is currently paraphyletic, but there are only two representatives for which we currently have molecular data.Additional sequences of Heterocotyle but also currently unrepresented taxa, Heliocotyle, Malalophus and Septesinus are required to provide a greater understanding of the evolutionary relationships within this group.
Heterocotylinae is amended in exclusion of Neoheterocotyle Hargis, 1955, Spinuris Doran, 1953, and Nonacotyle pristis Ogawa, 1991, all of which have prominently projecting, often spiculate dorsal haptoral sclerites.This exclusion also sees the removal of taxa with numerous anterior gland-duct openings, and those with four pairs of ventral pits in the anterior head region.The amended subfamily includes members with (a) rounded non-or partially sclerotised dorsal haptoral accessory structure(s) from myliobatiform stingrays and torpediniform electric rays and excludes monocotylids of rhinopristiform shovelnose rays.
Dasyatis chrysonota from South Africa is a new host record for Het.pastinacae.The discovery of Het.pastinacae on D. chrysonota rejects the hypothesis of Neifar, Euzet and Ben Hassine (2000) that Heterocotyle species are strictly host-specific and that they can be used to discriminate host species.Bullard et al. (2021) referred to the putative host-specificity dogma, evident in the monocotylid literature, suggesting that host-specificity in the family is probably less strict than has previously been considered.Indeed, this dogma is merely the result of the lack of sampling resolution across an extensive potential host diversity, much of which remains unexplored.The South African localities for Het.pastinacae, Hel.kartasi, and My.pteromylaei constitute new locality records for these species.

Troglocephalinae Vaughan n. subfam.
Diagnosis.Anterior head region arrow-shaped or rounded, with numerous single, distinct gland-duct openings along its anterolateral and lateral margins, not continuous along its entire posterior margin.Gland-duct openings connected to glands via network of inconspicuous gland ducts.Two anterolateral and one anteromedial gland usually present in the head region.Four pairs of ventral pits present in anterior head region.Eyespots present.Pharynx muscular, round, or ovoid.Intestinal caeca non-diverticular, non-confluent posteriorly.Ovary simple or lobed, positioned anterior to, or ventrally over single large ovoid, round, or lobed testis.Ovarian branch loops or does not loop right intestinal caecum.Vaginal pore armed or unarmed.Vagina single, with sclerotised or unsclerotised walls.Seminal receptacle single or bipartite.Oötype with ascending limb.Seminal vesicle a simple inflation from vas deferens.large musculo-glandular ejaculatory bulb present.Sclerotised male copulatory organ with or without accessory piece, with or without muscular sheath, without accessory filament; male copulatory organ significantly reduced in some species.Common genital pore armed or unarmed.Haptor roughly circular with

Remarks
Troglocephalus was chosen as the type-genus for the new subfamily because it was the original representative taxon to be excluded from all known subfamilies and was relegated to enigmatic status in the revised classification of the family by Chisholm et al. (2001a).The proposal of the new subfamily sees the inclusion of Anoplocotyloides and Mehracotyle insolita, transferred from Dasybatotreminae, Spinuris, Neoheterocotyle and Nonacotyle pristis from Heterocotylinae, and the inclusion of Troglocephalus rhinobatidis (previously incertae sedis).The current molecular phylogeny supports the separation of the representatives of the subfamily that have been sequenced, from Heterocotylinae, and from P. pelagica, the sole representative of Dasybatotreminae, further supporting the morphological hypothesis for the subfamily (Fig. 1).Currently, Anoplocotyloides, Mehracotyle, Nonacotyle, Timofeevia, and Spinuris are not represented in the molecular phylogeny, which is unavoidable and unfortunate, because a greater understanding of the relationships between these troglocephalines will ultimately result from the inclusion of their representative sequences.All these troglocephaline species are united by the morphology of the anterior head region and are currently only known from rhinopristiform shovelnose rays.
Some minor historic errors exist in the literature regarding the accuracy of some morphological features of representatives of the new subfamily.For example, Vaughan and Chisholm (2010b) illustrated and described what they considered the anterolateral glands in Ne. robii Vaughan & Chisholm, 2010; however, these illustrated structures are likely ganglia associated with anterior sensory cells, given that they do not 'open' to the ventral surface of the haptor as gland-duct openings do.The sub-anterolateral and lateral glands, depicted as close together in this species, are likely not separate, and are the true anterolateral glands.Nitta (2019) labelled the anterior glandduct openings along the margin of Ne. quadrispinata Nitta, 2019 as the anteromedian gland, which is a misnomer, where similarly, the anterior gland-duct opening of the anterior head glands were historically referred to as head organs; they are neither glands or organs (see Chisholm and Whittington 1996 for discussion).Chero et al. (2018) discussed Anoplocotyloides species as having marginal haptoral papillae.These marginal haptoral papillae are extensions from the outer-ring septum of the haptor (see Chisholm et al. 1995), and are only present in Clemacotyle, Dendromonocotyle and Monocotyle species (Monocotylinae), Dasybatotrema species and P. pelagica (Dasybatotreminae).The 'marginal haptoral papillae' considered by Chero et al. (2018) may stem from the descriptions of Anoplocotyloides chorrillensis Luque & Iannacone, 1991, which included small, rounded extensions of the marginal membrane that accommodate the 14 marginal hooks as marginal papillae.Given the definition of this character by Chisholm et al. (1995), marginal haptoral papillae are absent in Anoplocotyloides (see also Young 1967).The presence of an accessory filament on the male copulatory organ for A. papillatus (see Chisholm et al. 1995) is ◂ erroneous, possibly originating from the mistranslation of Bravo-Hollis (1969), where the male copulatory organ is described as surrounded by a delicate tubular membrane.No mention of an accessory filament is given in Doran (1953), Bravo-Hollis (1969), or Young (1967).Observations of the four pairs of ventral pits on the anterior head region were unclear in Anoplocotyloides chorrillensis and Spinuris species due to the level of staining employed, or the quality of presentation of this region on the slides; however, this character is clearly visible in A. papillatus, and the remaining characters of the new subfamily warrant the inclusion of Spinuris species.
Including the two new species into the original Dasybatotreminae classification would have resulted in yet another amendment of that subfamily to include additional ambivalent character states and the avoidance of resolving clear issues in Dasybatotreminae and Heterocotylinae.This would have resulted in Dasybatotreminae, as originally classified, becoming a dumping ground for clearly morphologically diverse taxa not accommodated in other subfamilies.
DNA reference sequences: two identical ribosomal 28S DNA reference sequences (962 bp) representing Scuticotyle cairae n. sp. are deposited in GenBank under accession numbers OR351733 and OR351734.The result from the BlastN search (01.04.24) resulted in no identical or close hits.The p-distances (Supplementary file S2) show a difference of 60 nucleotides or greater between S. cairae n. sp. and all other monocotylid taxa.The ML-analyses clearly demonstrate the placement of S. cairae n. sp. as a new species representing a separate genus within the Troglocephalinae (Fig. 1).

Remarks
The proposal of Scuticotyle is supported by the 28S ML-analyses and the combination of morphological features of the body-proper, the sclerotised male copulatory organ, and the vagina.Scuticotyle differs from all representatives of Troglocephalinae by the presence of the posterior shield-like projection of the body-proper (Fig. 2A), and the unique morphology of the male copulatory organ, which is reduced to thinly sclerotised adjacent walls exiting the musculo-glandular ejaculatory bulb and is not surrounded by a muscular sheath.The thinly sclerotised nature of these walls was assessed by proteolytic digestion and is confirmed in the staining using Gomori's Trichrome.The reduction of the male copulatory organ is peculiar; however, the presence of a field of papillae near the vaginal pore on the ventral tegument, suggests that this species likely produces external spermatophores, perhaps similar to those described for the microbothriid Dermopristis cairae Kearn, Whittington & Evans-Gowing, 2011, which are attached to the ventral tegument near the vaginal pore (Kearn et al. 2011).No spermatophores were observed in our specimens but spermatophores are known from this subfamily, from Ne. rhynchobatis (Chisholm and Whittington 1997).Scuticotyle is most similar to the genera Troglocephalus, and Brancheocotyle n. gen., all of which have seven radial haptoral loculi, no dorsal haptoral accessory sclerites, and a bipartite seminal receptacle (see Young 1967;Neifar et al. 2002 for this detail in Troglocephalus and Mehracotyle, respectively).The sclerotised male copulatory organ of Brancheocotyle n. gen. is also void of a muscular sheath, but this feature is present in Mehracotyle and Troglocephalus.The sclerotised male copulatory organ length of Scuticotyle, Brancheocotyle n. gen.and Mehracotyle is short, and longer in Troglocephalus.The vagina of Scuticotyle is a simple, inconspicuous, narrow, non-musculo-glandular tube but is musculo-glandular in Brancheocotyle n. gen.and Troglocephalus.The vagina is apparently missing in Mehracotyle (Neifar et al. 2002).It is possible that the vagina is present in Mehracotyle, and if so, that it too is a simple, inconspicuous tube, because this species has a seminal receptacle that was confirmed by Neifar et al. (2002) to contain sperm.Neither Scuticotyle nor Mehracotyle have a septal ridge along any of the haptoral septa.Additionally, Scuticotyle has fewer anterior head region glandduct openings (twelve or thirteen on either side) than Mehracotyle (twenty-six to thirty-three on either side; Neifar et al. 2002) and Troglocephalus (fifteen to twenty on either side) but more than Brancheocotyle n. gen (six or seven on either side).Mehracotyle is the only member of the subfamily with an ovary that does not loop the right intestinal caecum.The radial loculi are approximately of equal size in Scuticotyle, and Brancheocotyle n. gen.but the posterior haptoral loculus of Mehracotyle is notably much larger than any of its other haptoral loculi, and its posterolateral loculi are larger than the anterior and anterolateral loculi.Troglocephalus has dendritic structures within the marginal membrane of the haptor and an accessory piece associated with the hamulus, both absent in S. cairae and the other species.

Brancheocotyle n. gen.
Generic diagnosis: Anterior region of body with three anterior glands; anteromedian gland and anterolateral glands.Numerous large, singular, pad-like marginal anterior gland-duct openings present.Eight anterior ventral pits present.Paired anterior dorsal pits present.Granulated eyespots present.Haptor roughly circular with one central and seven peripheral loculi.Septal ridge present.Haptoral papillae absent.Septal or dorsal haptoral accessory sclerites absent.Marginal membrane present.Fourteen marginal hooklets distributed in the marginal membrane.Hamuli robust with reduced superficial root and long, thick dorsoventrally compressed deep root with deep longitudinal grooves, without accessory piece.Testis ovoid, medial.Musculo-glandular ejaculatory bulb present.Sclerotised male copulatory organ present, without accessory piece, accessory filament, or muscular sheath.Ovary with radial lobes overlapping testis ventrally.Ovarian branch loops around right intestinal caecum.Caecum without diverticula; non-confluent posteriorly.Unarmed vagina with conspicuous, unsclerotised muscular inner walls and surrounded in radial glandular tissue.Oötype straight.Common genital pore unarmed.Gill parasites of Rhinobatidae.Etymology: Named for the gill microhabitat, after the Latin branchiae for gills.Type and only species: Brancheocotyle imbricata n. sp.
DNA reference sequences: two identical ribosomal 28S DNA reference sequences (966 bp) representing Brancheocotyle imbricata n. sp. are deposited in GenBank under accession numbers OR351735 and OR351736.The result from the BlastN search (01.24.24) resulted in no identical or close hits.The p-distances (Supplementary file S2) show a difference of 70 nucleotides or greater between B. imbricata n. sp. and all other monocotylid taxa.The ML-analyses clearly demonstrate the placement of B. imbricata n. sp. as a new species representing a separate genus within the Troglocephalinae (Fig. 1).Type-host, localities and microhabitat as for S. cairae.Etymology: The species is named for the unique overlapping nature of the testis and ovary.The Latin word for overlapping is imbricata.Specimens: AHC 37059 (holotype); AHC 37060-37070 (11 paratypes); AHC 37021 (6 vouchers); AHC 37072 (3 digests).ZooBank registration: urn:lsid:zoobank.org:act:CC299745-AA48-4C75-9047-C59B889B6B0D.

Remarks
The proposal of Brancheocotyle is supported by the ML-analyses of the two 28S sequences, and the combined morphology of the ovary that has radial lobes situated ventrally directly over the testis, the musculoglandular morphology of the vagina, the morphology of the male copulatory organ, and the nature of the hamulus, which is reminiscent of the monotypic Cathariotrema selachii, having a reduced and irregularly truncated superficial root and a compressed, broad deep root with longitudinal grooves.Recently, C. selachii was redescribed by Bullard et al. (2021) demonstrating some morphological variability of isolates from different host species.Cathariotrema selachii is a member of Cathariotrematinae and differs markedly to Brancheocotyle in the general morphology of the anterior head region, the haptor, and has paired vaginal pores.Morphologically, the most similar genera are Mehracotyle, Scuticotyle, and Troglocephalus, all of which have seven peripheral haptoral loculi, no dorsal haptoral accessory structures, and a bipartite seminal receptacle.The hamuli of Mehracotyle, Scuticotyle, and Troglocephalus all have a narrow deep root, and the superficial root of Mehracotyle and Scuticotyle is well-developed.The superficial root of Troglocephalus is considered reduced (Chisholm et al. 1995) but it is not irregularly truncated.Troglocephalus also has a hamular accessory piece that is associated with the hook portion of the hamulus, absent in the other taxa.The ovary of Brancheocotyle is unique in the Monocotylidae in that it is proximally lobed and lies directly ventral over the testis.A lobed ovary is present in other monocotylids, notably Peruanocotyle species (Dasybatotreminae); however, the ovary Peruanocotyle does not overlap with its four testes.Mehracotyle and Scuticotyle have a simple, unlobed ovary.Troglocephalus has an unlobed, weakly sinuous or irregular-shaped proximal portion of the ovary.The ovary of Mehracotyle does not loop the right intestinal caecum but it does in Brancheocotyle, Scuticotyle, and Troglocephalus.The vagina of Brancheocotyle is strongly muscular and is surrounded by conspicuous glandular tissue.The vagina of Troglocephalus is also musculo-glandular; however, the vagina is not musculo-glandular in Scuticotyle (see the remarks section for this taxon for the comparative discussion on the purported absence of a vagina in Mehracotyle).The sclerotised male copulatory organ of Brancheocotyle, Scuticotyle and Mehracotyle is short, and is surrounded by a muscular sheath in Mehracotyle and Troglocephalus only.Additionally, a single, straight haptoral septal ridge is present in Brancheocotyle and Troglocephalus (confirmed in the present study) but is absent in Mehracotyle and Scuticotyle.The latter genus includes an additional shield-like structure extending beyond the posterior portion of the body-proper, absent in all other taxa.The marginal hooklets of Brancheocotyle appear to be larger and more robust than those of the other taxa.Dasybatotreminae Bychowsky, 1957.Revised diagnosis.Anterior head region roughly circular in shape, with numerous, parallel radiating grooves or parallel radiating rows of multiple small gland-duct openings, present along the majority of the circumferential margin.Anterior-most part of head region with or without anterior notch.Eight anterior ventral pits absent.Eyespots present or absent.Pharynx muscular, ovoid.Intestinal caeca diverticular or non-diverticular, non-confluent posteriorly.Male copulatory system with musculo-glandular ejaculatory bulb with or without bipartite internal portion of seminal vesicle.sclerotised male copulatory organ, with or without accessory piece, without accessory filament.One or four ovoid testes present.Ovary positioned anterior to testis/testes simple or proximally lobed; distal branch highly convoluted, or not, looping right intestinal caecum.Vagina single; vaginal wall sclerotised or not; vaginal pore unarmed.
Oötype with ascending limb only.

Remarks
The 'short' nature of the deep hamular root in Dasybatotreminae is represented only in P. chisholmae.It is elongate in all other members of the subfamily.Chisholm et al. (1995) included that the length of the hamulus deep root for the group, was greater than half that of the radial haptoral septa, and that the superficial root was either reduced or well-developed.These authors also included that the absence of a sinuous haptoral septal ridge, the absence of septal, papillary, and dorsal haptoral accessory structures (either sclerotised or not), and a non-diverticular ceacum, were typical in this group.Peruanocotyle species have very small hamuli, which prompted Chero et al. (2018) to amend the description of this character state to the deep root length being greater than half the length of the radial septa or shorter than the width of the marginal membrane.Chero et al. (2018) also included the presence of the diverticular caecum, and the presence of four testes, for Peruanocotyle.All representatives of Dasybatotreminae have the characteristic radiating arrangement of parallel rows of grooves, or gland-duct openings around nearly the entire circumference of the anterior head region (see representations in Price 1938for D. dasybatis, Timofeeva 1983for D. spinosum and T. rajae, Chero et al. 2018for P. chisholmae, and Ruiz-Escobar et al. 2022 for P. pelagica).The morphology of these parallel radiating grooves or gland-duct openings is unique in this group.The stained voucher specimen HWML 17119_17164 clearly demonstrates that these parallel radiating rows in D. dasybatis are made up of many very small, separate individual openings (Fig. 7).These differ significantly in form from the large, conspicuous, singular, marginal glandduct openings of Troglocephalinae representatives.Although Chero et al. (2018) and Ruiz-Escobar et al. (2022) did not evaluate the glandular nature of these grooves in Peruanocotyle species, Chero et al. (2018) did detail the presence of three large head glands in P. chisholmae, which are associated with gland-duct openings in other representative monocotylid subfamilies.Given that there are no other structures present that would discount these radiating grooves or rows of gland-duct openings as glandular in nature, we consider them analogous to the gland-duct openings in other members of the family.Chero et al. (2018) included an unarmed common genital pore character, and the presence or absence of an accessory filament associated with the male copulatory organ in their revised subfamily diagnosis for Dasybatotreminae; however, the type-genus, Dasybatotrema contains species with an armed common genital pore (Chisholm et al. 1995), and the presence of an accessory filament was based on its erroneous inclusion for A. papillatus by Chisholm et al. (1995), now a representative of Troglocephalinae.A bipartite portion of the seminal vesicle, internal within the ejaculatory bulb, was described by Chero et al. (2018) for P. chisholmae and is also demonstrated by Timofeeva (1983) for T. rajae.Table 2 Estimates of net evolutionary divergence between sequences for subfamilies, Monocotylidae sp.incertae sedis,

and the outgroup
The number of base differences per sequence from estimation of net average groups of sequences are shown.The rate variation among sites was modelled with a gamma distribution (shape parameter = 1).This analysis involved 79 nucleotide sequences.All ambiguous positions were removed for each sequence pair (pairwise deletion option).
There were a total of 1236 positions in the final dataset.Evolutionary analyses were conducted in MEGA11 (Tamura et al. 2021) (pers. comm.).We investigated the historic, meticulous field notes and data originating from the study of Chisholm et al. (2001a).Of all the taxa listed in Chisholm et al. (2001a), this species was the only one for which we could not find any confirmatory morphological identity data, or data verifying the identity or collection date of the purported host species.It is clear from the current phylogeny that AF348362 represents a member of the Monocotylidae, but that it is not a Neoheterocotyle species (Fig. 1).The transferral of Neoheterocotyle to the new subfamily addresses the historic polyphyly of the Heterocotylinae.The representatives of Heterocotylinae, as amended, reduces the known host association of the group to gill parasites of myliobatiform and torpediniform rays.Heterocotyle is however paraphyletic within Heterocotylinae with the inclusion of the new sequence for Het.tokoloshei (Fig. 1).Heterocotyle capricornensis is currently represented as a separate taxon to the rest of the heterocotylinids within the subfamily (Fig. 1); however, morphologically, this species is still considered a well-supported representative of the subfamily.This species was admittedly problematic in both morphological and molecular phylogenies presented by Chisholm and Whittington (1996) and Chisholm et al. (2001a).In the morphological phylogeny of the genus (Chisholm and Whittington 1996), Het.capricornensis was either placed in an unresolved trichotomy with all other Heterocotyle species, or grouped with the outgroups (Ne.rhinobatidis, Nonacotyle pristis, Potamotrygonocotyle tsalickisi Mayes, Brooks &Thorson, 1981, andSpinuris lophosoma Doran, 1953).This resulted from the unique combination of two characters states in Het.capricornensis: a single sinuous haptoral ridge, and a male copulatory organ without an accessory piece (Chisholm and Whittington 1996).In the molecular phylogeny of Chisholm et al. (2001a), Het.capricornensis was presented as a separate taxon, sister to Decacotylinae, or in a polychotomy between a group represented by Troglocephalus and three Neoheterocotyle species, and a group representing Decacotylinae.Chisholm et al. (2001a) urged the inclusion of additional Heterocotyle species into future molecular phylogenies to evaluate the validity of Heterocotyle.The inclusion of Het.tokoloshei provides only limited additional resolution, thus we cannot comment on the validity of the genus; however, Chisholm and Whittington (1996) did describe additional unique characters for Het.capricornensis: a tri-lobed testis, and a distal ovarian loop, anterior to the initial caecal ovarian loop.Whether these unique characters reflect a generic difference is debatable, although the current phylogenetic position of the two Heterocotyle species might support this hypothesis.Decacotylinae was represented as a sister group to Calicotylinae, Cathariotrematinae, and Merizocotylinae in Bullard et al. (2021); however, in the current molecular phylogeny, Decacotylinae is unresolved, representing Decacotyle as paraphyletic and as basal to all other members of the family (Fig. 1).
The Loimoinae was formally incorporated into the Monocotylidae by Chero et al. (2021) with the inclusion of Loimopapillosum pascuali Chero, Cruces, Sáez, Oliveira, Santos & Luque, 2021.In the earlier molecular phylogeny of Boeger et al. (2014), who opted to provisionally retain Loimoidae Price, 1936, this taxon, represented by Loimosina Manter, 1944sp. (= Loimosina wilsoni Manter, 1944;see Dalrymple et al. 2022) was considered a sister group to Tr. rhinobatidis (previously as incertae sedis) and Ne.rhinobatis (Monocotylidae sp.incertae sedis; for the previously polyphyletic Heterocotylinae).Moreover, Boeger et al. (2014) considered the ventral pits of the anterior head region, which are present in Loimos MacCallum, 1917 and Loimosina as a synapomorphy for a group representing Loimoidae (Loimosina wilsoni), Tr. rhinobatidis and Ne.rhinobatis (Monocotylidae sp.incertae sedis).In our phylogenetic analysis, the presence of ventral pits as a synapomorphy is also the most parsimonious hypothesis, where this character is secondarily lost in Dasybatotreminae (represented by Peruanocotyle), but also in Loimopapillosum Hargis, 1955. Boeger et al. (2014) also suggested a possible phylogenetic relationship between Me. insolita (for Dasybatotreminae) and Loimoidae (Loimosina wilsoni) based on the lack of the ovarian branch looping the right intestinal caecum.Chero et al. (2021) also recognised the possible phylogenetic relationship between Loimos, Loimosina, Me. insolita (for Dasybatotreminae) and A. papillatus (for Dasybatotreminae), based on the ventral pits and the non-looping ovarian branch.However, their inclusion of Loimopapillosum pascuali represented Loimoinae as they defined it, as paraphyletic, with Loimopapillosum pascuali as a sister group to Het. capricornensis (Heterocotylinae).Dalrymple et al. (2022) came to the same conclusion regarding the paraphyly of the loimoids, opting to take a more conservative approach to the group, refraining from classifying them as Loimoidae or Loimoinae until additional sequences could be included of representative members.The sequence for Loimos sp. is the first representative sequence for this genus to be included in a molecular phylogeny of the family (Fig. 1), and it groups together with Loimosina with high support.Loimopapillosum was reported previously as a representative of the Loimoinae (Chero et al. 2021) but this was not supported in their presented phylogenies based on 28S rDNA sequences.Loimopapillosum grouped together with Loimosina in the 18S phylogeny of Chero et al. (2021); however, this phylogeny was based on a very restricted number of taxa.Dalrymple et al. (2022) included Loimopapillosum in a larger phylogeny, also based on 28S, and clearly demonstrated that Loimopapillosum and Loimosina were not part of the same group.Similarly, Loimopapillosum is not supported in the current phylogeny as a representative of Loimoinae.We agree with Boeger et al. (2014) and Dalrymple et al. (2022) that additional work is required for this group before a decision can be made on assigning members of the group to only a single subfamily.Loimopapillosum is morphologically very different from Loimos and Loimosina, and there are at least 161-169 pairwise nucleotide differences between Loimopapillosum pascuali and Loimosina wilsoni, and 161 between Loimopapillosum pascuali and Loimos sp.; there are 64-73 nucleotide differences between Loimosina wilsoni and Loimos sp.(see Supplementary file S2).Based on the morphological differences between Loimopapillosum species and the other members of the group, and the clear separation of Loimopapillosum in the current phylogeny, Loimopapillosum pascuali is considered incertae sedis.Additional future data from new specimens of Loimopapillosum might justify the consideration of a separate subfamily to accommodate it.
Monocotylinae is not supported as a monophyletic group in the current phylogeny (Fig. 1).Similarly, the phylogenetic analysis of Bullard et al. (2021) and Dalrymple et al. (2022) presented low support for the Monocotylinae.The separation of the group containing Dendromonocotyle Hargis, 1955 andClemacotyle Young, 1967 from the group containing Monocotyle Taschenberg, 1878 provides support for reinstating Dendromonocotylinae Hargis, 1955.This separation reflects the host microhabitats, where Monocotyle species are parasites of the gill tissue, and Dendromonocotyle and Clemacotyle are parasites of the external skin surface and gill cavity, respectively.Chisholm et al. (1995) were aware of the morphological support for Dendromonocotylinae; however, its formal recognition would have reduced Monocotylinae to the single genus based on homoplasy (see also Chisholm et al. 2001a); therefore, these authors chose to assign Dendromonocotyle and Clemacotyle to Monocotylinae.This decision was supported in the molecular phylogeny of Chisholm et al. (2001a), representing a monophyletic Monocotylinae.The relationship between Dendromonocotyle and Clemacotyle is still unresolved, and additional sequences but also a close re-evaluation of morphological characters within this subfamily are needed before an alternative treatment is proposed for Monocotylinae.
The current study includes a significant contribution to the known diversity of the Monocotylidae off South Africa and the phylogenetic resolution within the family.The inclusion of B. imbricata and S. cairae, C. selachii, Het.pastinacae, Hel.kartasi, and My.pteromylaei, doubles the previous number of described monocotylid species (Vaughan et al. 2021) to twelve, and increases the number of representative genera from this region from four to nine.younger DBV sought the advice of the late A/Prof.Ian Whittington on where to begin with this study.We would like to thank Prof. Janine Caira for the donation of monogeneans during her sampling on board the RV Africana in 2010.We are grateful to Dr Pauline Narvaez for her kind assistance with the English translation of French manuscripts, to Dr Anna Phillips and Dr Yolanda Villacampa, and the Scientific Imaging Lab of Dr Scott Whittaker at the National Museum of Natural History, Smithsonian Institution, USA for the full Z-stack imagery of Anoplocotyloides chorrillensis, Dr Marcelo Knoff and Dr Daniela de Almeida Lopes of the Oswaldo Cruz Institute, Brazil for the photomicrographs of Peruanocotyle chisholmae, and to the late Eileen Harris, Mr Jon Ablett and Dr Lauren Hughes of the British Natural History Museum for access to Mehracotyle insolita.Dr Rob Leslie provided valuable assistance with the taxonomic identification of hosts species on board the RV Africana.We are grateful to Professor Marcus Vinicius Domingues, Federal University of Pará, Brazil, for access and permission to use his photographic library.DBV would also like to thank the captain and crew of the RV Africana for hosting him during multiple research voyages, Ms Judy Couper, and Mr Wayne Pederick from the School of Health, Medical and Applied Sciences, Central Queensland University, Australia for providing access to the Nikon Eclipse 200, and Prof. Ash Bullard for commentary on earlier aspects of the manuscript.This paper is dedicated to the memory of Sheilagh Vaughan who passed away in October 2022.Thanks to Saima Nasrin Mohammad, Norwegian Veterinary Institute, Norway, for help with PCR and DNA sequencing.
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Fig. 1
Fig. 1 Molecular phylogenetic analyses of the Monocotylidae assessed by maximum likelihood inference.The best fit tree is shown.Branch support assessed by ultrafast bootstrap (UFBoot2).

Fig. 5
Fig. 5 Brancheocotyle imbricata n. gen.et sp. A. Whole mount ventral view.B. Hamulus, demonstrating variation in length of handle in the size range of adult specimens observed.

Fig. 7
Fig. 7 Portion of anterior head region of Dasybatotrema dasybatis voucher HWML 17719_17164, demonstrating the detail of the parallel rows of gland-duct openings radiating around the margin.Scale bar = 100 µm.Photomicrograph taken by Professor Marcus Vinicius Domingues.

Table 1
Taxa and their 28S rDNA sequence accession details used in the molecular phylogeny (new sequences in bold text) Taxon

Acroteriobatus annulatus Off Cape Agulhas, South Africa OR351737, OR351738 Present study
with pairs of few (usually three), small anterolateral gland-duct openings on the anterolateral margin, and usually one pair of smaller, indistinct anteromedial gland-duct openings near the anterior margin.Two 1 Dalrymple et al. (2022) considered Loimosina sp.WAB-2014 of Boeger et al. (2014) to be representative of L. wilsoni 2 Dalrymple et al. (2022) suggested that L. parawilsoni Bravo-Hollis, 1970 is a junior synonym of L. wilsoni 3 Considered in the present study as Monocotylidae sp.incertae sedis ally short, narrow, but elongated and broad in Electrocotyle.Fourteen marginal hooks distributed in the marginal membrane.Single straight, slightly sinuous, or sinuous, or double-sinuous septal ridge present.Numerous longitudinal sinuous ridges on ventral surface of peripheral loculi present or absent.Marginal haptoral papillae absent.Septal sclerites absent.