NOVELTY AND PHYLOGENETIC RELATIONSHIPS WITHIN THE SERENDIPEIDAE (CESTODA: "TETRAPHYLLIDEA").

Nanoduplicibothrium n. gen. is erected for the subgroup containing the smallest members of the "tetraphyllidean" family Serendipeidae with bothridia fused lengthwise in 2 pairs that lack both a distinct row of posterior loculi and a cephalic peduncle. Two new species in this genus are described. These are Nanoduplicibothrium leanneae n. gen. n. sp. from Rhinoptera bonasus off South Carolina and Nanoduplicibothrium megaphallum n. sp. from Rhinoptera jayakari off Mozambique. Two species currently assigned to Duplicibothrium are transferred to the new genus as Nanoduplicibothrium paulum n. comb and Nanoduplicibothrium jillae n. comb. and the diagnosis of Duplicibothrium is emended so that it aligns with the revised membership of the group. Duplicibothrium bilai n. sp. is also described from R. jayakari off Mozambique. The description of these species provides formal names for 3 species included in previously published molecular phylogenetic work under the provisional names Duplicibothrium n. sp. 2, Duplicibothrium n. sp. 4, and Duplicibothrium n. sp. 5, respectively. Erection of the new genus substantially reduces the number of instances of congeners in the family parasitizing the same host species because in most instances the pairs of species now represent 1 species each in Nanoduplicibothrium and Duplicibothrium. Sequence data for the D1-D3 region of the 28S rDNA gene were generated for Serendip for the first time from an undescribed species from Aetomylaeus asperrimus collected off Panama. This finding also expands the known host associations of the Serendipeidae beyond the Rhinopteridae to include a species of Myliobatidae. A maximum-likelihood phylogenetic analysis of all species of serendipeids for which data for the D1-D3 region of the 28S rDNA gene are available confirms the reciprocal monophyly of Nanoduplicibothrium, Duplicibothrium, and Serendip. The phylogenetic placement of the fourth genus in the family-the monotypic Glyphobothrium-remains to be determined.

Phylogenetic relationships, host associations, and three new species of a poorly known group of "tetraphyllidean" tapeworms from elasmobranchs.

This paper aims to expand understanding of a poorly known group of cestodes that parasitize an intriguingly diverse suite of elasmobranchs. The group's three currently described members (i.e., Pentaloculum macrocephalum, Pentaloculum hoi, and Zyxibothrium kamienae) parasitize an electric ray, a carpet shark, and a skate, respectively. Pentaloculum grahami n. sp. is described from a second genus of carpet shark, specifically Parascyllium collare, in Australia. Zyxibothrium duffyi n. sp. and Zyxibothrium healyae n. sp. are described from the deep-sea skates Brochiraja asperula and Brochiraja spinifera, respectively off New Zealand. The three new species share distinctive bothridia that bear a small number of large, circular, facial loculi and lateral bands of vitelline follicles that converge posterior to the ovary-features which are found in all other members of these genera. Zyxibothrium healyae n. sp. is unique in possessing three, rather than four or five, facial loculi. Zyxibothrium duffyi n. sp. possesses a combination of five facial loculi and vitelline follicles that stop short of the anterior margin of the proglottid. Pentaloculum grahami n. sp. is the largest member of the group with the greatest number of proglottids. Based on striking similarities in scolex morphology, Pentaloculum and Zyxibothrium have been hypothesized to belong to a distinct subgroup of "tetraphyllideans" provisionally designated as Clade 1. Based on sequence data for the D1-D3 region of the 28S rDNA gene generated for species of Zyxibothrium for the first time, we confirm the reciprocal monophyly of both genera as well as the monophyly of Clade 1 and its status as a distinct lineage among the "Tetraphyllidea". This work also suggests that the presence of five facial loculi is homoplasious given this character state is found in members of both genera. The new species expand the host associations of Clade 1 to include additional skate and carpet shark genera. Moving forward we would expect to find additional members of this group parasitizing other species of parascyliid carpet sharks as well as other species of the rajid genus Malacoraja and the arhynchobatid genus Brochiraja. Here we have doubled the number of described species in the taxon referred to as Clade 1 while simultaneously expanding our understanding of the morphology and anatomy of its members. This additional information will help inform the ultimate revision of the ordinal classification of the cestodes to address the highly polyphyletic nature of the order "Tetraphyllidea" as it is currently configured.

Novelty and phylogenetic affinities of a new family of tapeworms (Cestoda: Rhinebothriidea) from endangered sawfish and guitarfish.

The parasites of hosts of conservation concern are often poorly known. This is the case with the iconic group of elasmobranchs known as the sawfish of the genus Pristis, all four species of which are considered as Endangered or Critically Endangered by the International Union for Conservation of Nature (IUCN, Switzerland). Examination of cestodes from three species of sawfish (Pristis pristis, Pristis clavata, and Pristis zijsron) in Australia and one of their close relatives, the also critically endangered widenose guitarfish, Glaucostegus obtusus, in India, collected over the past 25 years, yielded four new species of tapeworms which are described herein. All four belong to the previously monotypic Mixobothrium; the diagnosis of the genus is revised to accommodate the new species. Among the new taxa is a species that had been included in previous molecular phylogenies but whose identity and affinities within the order Rhinebothriidea, and thus also its familial placement, were unclear. This species exhibits the morphological features of Mixobothrium and thus its identity is, at long last, revealed. Sequence data generated for the 28S rDNA gene for three of the new species, as well as an additional new but yet undescribed species from Pristis pectinata from Florida (USA), confirms the uniqueness of this group among the rhinebothriideans. The new family Mixobothriidae is established to house these taxa. The members of this family differ from all but one of the five other families of rhinebothriideans in lacking apical suckers on their bothridia. They are also distinctive in that their bothridia are divided into three regions. The anterior and posterior regions have similar locular configurations to one another and differ from the locular configuration of the middle region. As a consequence, the bothridia are symmetrical along both their vertical and horizontal axes. We predict that a focus on species of guitarfish in the genus Glaucostegus will be the most productive approach for discovering additional diversity in this family of cestodes.

Diversity and phylogenetic relationships of 'tetraphyllidean' Clade 3 (Cestoda) based on new material from orectolobiform sharks in Australia and Taiwan.

In an effort to expand knowledge of Clade 3-one of the ten clades that compose the non-monophyletic order 'Tetraphyllidea' all current members of which parasitise orectolobiform sharks-we targeted species of orectolobiform sharks that had not previously been examined for 'tetraphyllidean' cestodes. That work led to the discovery of three new species off Australia and Taiwan. Ambitalveolus gen. n. was erected to accommodate these species. Ambitalveolus costelloae gen. n. et sp. n., Ambitalveolus kempi sp. n., and Ambitalveolus penghuensis sp. n. differ from one another in scolex size, genital pore position, and number of marginal loculi, proglottids, and testes. Among 'tetraphyllideans', the new genus most closely resembles the two other genera in Clade 3. It differs from Carpobothrium Shipley et Hornell, 1906 in lacking anterior and posterior flap-like extensions of its bothridia; instead, its bothridia are essentially circular. It differs from Caulopatera Cutmore, Bennett et Cribb, 2010 in that its vitelline follicles are in two lateral bands, rather than circum-medullary, and in that its bothridia bear, rather than lack, conspicuous marginal loculi. A key to the three genera in Clade 3 is provided. A phylogenetic analysis including new sequence data for one of the three new species of Ambitalveolus gen. n., the only species of Caulopatera, and all four described species and one undescribed species of Carpobothrium supports previously hypothesised close affinities between Caulopatera and Carpobothrium, with the new genus as their sister group. This is the first report of 'tetraphyllidean' cestodes from the orectolobiform shark family Brachaeluridae Applegate. The association of the new species with orectolobiform sharks is consistent with those of the other members of Clade 3. However, whereas species of Carpobothrium and Caulopatera parasitise members of the hemiscylliid genus Chiloscyllium Müller et Henle, species of Amitalveolus gen. n. parasitise members of the Brachaeluridae and Orectolobidae Gill.

Three new species of Duplicibothrium (Cestoda: 'Tetraphyllidea') from cownose rays in Senegal with a phylogenetic analysis of the genus.

Three new species of 'tetraphyllideans' in the family Serendipeidae are described from cownose rays off Senegal. Duplicibothrium jeannettae n. sp. and Duplicibothrium collosum n. sp. parasitize both Rhinoptera marginata and Rhinoptera peli. Duplicibothrium jillae n. sp. parasitizes R. marginata. Duplicibothrium jeannettae n. sp. and D. jillae n. sp. differ from one another and their described congeners in size, number of proglottids, and locular configuration. The generic assignment of D. collosum n. sp. was initially unclear because its scolex more closely resembles that of Serendip than Duplicibothrium. To help inform generic placement, sequence data were generated for the 28S rDNA gene (D1-D3 region) for the three new species as well as for Duplicibothrium minutum and Duplicibothrium n. sp. 2 from Rhinoptera bonasus off North Carolina, Duplicibothrium n. sp. 3 from Rhinoptera brasiliensis off Belize, Duplicibothrium n. sp. 4 and Duplicibothrium n. sp. 5 from Rhinoptera jayakari off Mozambique, and Duplicibothrium n. sp. 6 from Rhinoptera neglecta off Australia. Comparable data were obtained from GenBank for adults and larvae of Duplicibothrium collected from the Gulf of Mexico. The tree resulting from a maximum likelihood analysis (MLA) placed D. collosum n. sp. robustly within Duplicibothrium; the generic diagnosis is emended accordingly. This raises a question regarding the independence, and thus also validity, of the three genera of the Serendipeidae - a question that must await a molecular analysis that includes Serendip and Glyphobothrium. These results extend the hosts of Duplicibothrium to include R. brasiliensis, R. jayakari, R. marginata, R. neglecta and R. peli, and the distribution to include the western Pacific Ocean, eastern Atlantic Ocean, and western Indian Ocean (Zoobank Registration: 97BB5020-BFFF-4FEA-AE07-B4711D1110FC).

A revision of the Rhoptrobothriidae (Cestoda: Tetraphyllidea).

The Rhoptrobothriidae are one of the more enigmatic families of cestodes of elasmobranchs. Opinions on the taxonomic status of the familys three original genera (i.e., Myzophyllobothrium, Rhoptrobothrium, and Myzocephalus) have varied over the 115 years since they were erected. Some authors have considered all three valid, others have considered Rhoptrobothrium to be a synonym of Myzopyllobothrium or a genus inquirendum, yet others have considered Myzocephalus to be a synonym of the phyllobothriid genus Thysanocephalum. All three genera were established for specimens collected from eagle rays off Sri Lanka. The erection of Mixophyllobothrium for two specimens from a cowtail stingray off India three decades ago added additional confusion to the situation, with some authors considering it valid and others a synonym of Myzocephalus. These disagreements stem largely from differences in interpretation of the complex morphology of the scolex of members of these genera. Furthermore, with the exception of Rhoptrobothrium comprising four species, each genus is monotypic. All but Rhoptrobothrium has not been considered in detail for nearly a century, largely because of a lack of available material. The taxonomic status of these genera is assessed here based on light and scanning electron microscopy, and molecular data generated from new material collected from eagle rays off Indonesian and Malaysian Borneo, Japan, Sri Lanka, and Viet Nam. Morphological work indicates that the genera differ largely only in the degree of folding of the four remi that extend from the cephalic peduncle. A molecular phylogeny based on sequence data for the D1D3 region of the 28S rRNA gene, which include new data for eight specimens of four species, indicates that Myzophyllobothrium, Myzocephalus, and Rhoptrobothrium are not mutually monophyletic. The latter two genera and Mixophyllobothrium are considered synonyms of Myzophyllobothrium and five species are transferred to that genus. Myzophyllobothrium okamuri n. comb. is considered a species inquirendum. Myzophyllobothrium nagasawai n. sp. is described from Aetobatus narutobiei off Japan. Myzophyllobothrium narinari n. comb. is re-described based on newly collected cestodes from the type host and locality (i.e., Aetobatus ocellatus off Sri Lanka). Despite consisting of only a single genus, the family status of the group is retained in recognition of the unusual configuration of the scolex, which bears four biloculate bothridia and four remi extending from the cephalic peduncle. The ordinal placement of the family remains uncertain, but affinities with the Phyllobothriidea, rather than Tetraphyllidea are considered.

Two new species of Caulobothrium (Cestoda: "Tetraphyllidea") from the duckbill eagle ray, Aetomylaeus bovinus (Myliobatiformes: Myliobatidae), off Senegal with new insights on morphological features of the genus.

Two new species of the cestode genus Caulobothrium, collected from the duckbill eagle ray, Aetomylaeus bovinus, off Senegal, are described. Although postulated as sister taxa in an earlier molecular phylogenetic analysis, Caulobothrium multispelaeum n. sp. and Caulobothrium katzi n. sp., respectively, are among the smallest and largest members of the genus. The smaller species is unique among its congeners in possessing unusual medial longitudinal grooves along the dorsal and ventral surfaces of its strobila that develop into a tandem series of elliptical apertures on the posterior proglottids. The inner surfaces of these apertures stained positively with McManus' periodic acid Schiff in a manner similar to that seen in members of the distantly related lecanicephalidean genus Elicilacunosus. The larger species differs from its congeners in size, number of proglottids, and arrangement of bothridial loculi. Both new species were found to possess a small apical sucker on the anterior margin of each of their bothridia. Scanning electron microscopy (SEM) and frontal sections of a bothridium of Caulobothrium tetrascaphium suggests that this species also bears an apical sucker. Examination of the hologenophore of the species provisionally referred to as Caulobothrium n. sp. 5 in the earlier molecular analysis indicates it is conspecific with the recently described Caulobothrium pedunculatum, which was also determined to possess bothridial apical suckers. This leads us to suspect that this feature may be found to occur in all members of the genus. SEM of specimens of Caulobothrium for the first time indicates their bothridial surfaces are covered with filitriches of various sizes but lack spinitriches; spinitriches were seen only on the cephalic peduncle of C. katzi n. sp. The geographic distribution and host associations of Caulobothrium are expanded to include data now available for all species. The diagnosis of the genus is revised to include all of this information.

Three new species of 'tetraphyllidean' cestodes from an undescribed bamboo shark (Orectolobiformes: Hemiscylliidae) in Sri Lanka.

Examination of seven specimens of an undescribed species of bamboo shark (Orectolobiformes: Hemiscylliidae), currently referred to as Chiloscyllium sp. 1, from Sri Lanka yielded three new species of 'tetraphyllidean' cestodes, one each in the genera Carpobothrium Shipley et Hornell, 1906, Spiniloculus Southwell, 1925, and Yorkeria Southwell, 1927. Carpobothrium marjorieae Caira, Otto et Jensen sp. n. differs from its three valid congeners in total length, number of testes, and size of the apical sucker of its anterior bothridial flap. Like Carpobothrium eleanorae Koontz et Caira, 2016 it possesses spherical eggs with highly elongate bipolar filaments, but its eggs are conspicuously larger than those of the latter species. Spiniloculus akshayi Caira, Otto et Jensen sp. n. can be distinguished from its five congeners in total length and number of proglottids. Yorkeria sachiniae Caira, Otto et Jensen sp. n. differs from its 16 congeners in number of proglottids and testes, total length, terminal proglottid, cirrus sac, and cephalic peduncle length, hook size, genital pore position, and bothridial shape. The eggs of Y. sachiniae Caira, Otto et Jensen sp. n. are spindle-shaped and bear a single long, polar filament. All three species were found to exhibit microthrix patterns on their scolices like those of the majority of their congeners that have been examined to date with scanning electron microscopy. Microtriches on the distal surfaces of the bothridial pouches of a species of Carpobothrium were characterised for the first time; C. marjorieae Caira, Otto et Jensen sp. n. was found to possess a modified form of gladiate spinithrix on this surface. Among the seven members of the genus, this is the first species of Chiloscyllium Müller et Henle that has been found to host representatives of all three of these 'tetraphyllidean' genera. However, unlike most of its congeners, a representative of the non-hooked genus Scyphophyllidium Woodland, 1927 was not found infecting Chiloscyllium sp. 1.

Electron microscopy reveals novel external specialized organs housing bacteria in eagle ray tapeworms.

Nutritionally-based mutualisms with bacteria are known to occur in a wide array of invertebrate phyla, although less commonly in the Platyhelminthes. Here we report what appears to be a novel example of this type of association in two geographically disparate and phylogenetically distant species of tapeworms of eagle rays-the lecanicephalidean Elicilacunosus dharmadii off the island of Borneo and the tetraphyllidean Caulobothrium multispelaeum off Senegal. Scanning and transmission electron microscopy revealed that the grooves and apertures on the outer surfaces of both tapeworms open into expansive cavities housing concentrations of bacteria. This led us to reject the original hypothesis that these structures, and their associated mucopolysaccharides, aid in attachment to the host mucosa. The cavities were found to be specialized in-foldings of the tapeworm body that were lined with particularly elongate filitriches. Given tapeworms lack a gut and employ filitriches to assist in nutrient absorption, enhanced nutrient uptake likely occurs in the cavities. Each tapeworm species appeared to host different bacterial monocultures; those in E. dharmadii were coccoid-like in form, while those in C. multispelaeum were bacillus-like. The presence of bacteria in a specialized structure of this nature suggests the structure is a symbiotic organ. Tapeworms are fully capable of obtaining their own nutrients, and thus the bacteria likely serve merely to supplement their diet. Given the bacteria were also extracellular, this structure is more consistent with a mycetome than a trophosome. To our knowledge, this is not only the first evidence of an external symbiotic organ of any type in a nutritionally-based mutualism, but also the first description of a mycetome in a group of invertebrates that lacks a digestive system. The factors that might account for the independent evolution of this unique association in these unrelated tapeworms are unclear-especially given that none of their closest relatives exhibit any evidence of the phenomenon.

Three New Species of Rhinebothrium (Cestoda: Rhinebothriidea) from the Leopard Whipray, Himantura Leoparda, in Australia.

Examination of 4 specimens of the leopard whipray Himantura leoparda, a dasyatid stingray from northern Australia, led to the discovery of 3 new species of Rhinebothrium. Rhinebothrium leopardensis n. sp., Rhinebothrium nandoi n. sp., and Rhinebothrium ruhnkei n. sp. are described, increasing the diversity of the genus to 51 species globally. All 3 new species differ from their congeners in terms of testis number, proglottid number, loculus number, and size. With respect to one another, R. leopardensis n. sp. has bothridia that are weakly constricted at their centers and has a greater number of proglottids than the other 2 species (93-108 vs. 11-15, and 48-78, respectively). Rhinebothrium nandoi n. sp. is the smallest of the 3 species found in H. leoparda (3.6-5 vs. 10-15 mm and 10.1-15.8 mm in total length [TL], respectively) and bears bothridia that are constricted at their centers. Rhinebothrium ruhnkei n. sp. bears bothridia that are conspicuously constricted at their centers and has more testes than R. leopardensis and fewer than R. nandoi (7-10 vs. fewer than 7 and 21-33, respectively). Before this study, 56% (27 of 48) of Rhinebothrium species had been described from the freshwater river systems of South America and the marine waters surrounding South and North America. In contrast, despite the remarkably diverse nature of its batoid fauna, only 19 species were known from the Indo-Pacific region. Our work increases this number to 22, emphasizing the highly underestimated nature of Rhinebothrium diversity in this region of the globe. The discovery of these 3 new species was not unexpected, given the relatively poor status of our current knowledge of the cestode faunas of dasyatid stingrays in the Indo-Pacific region, and given the fact that it is common for a single batoid species to host 2 or more species of Rhinebothrium. Our results suggest that additional work on the cestode faunas of the batoids, especially dasyatids, from the Indo-Pacific region is likely to be highly productive in terms of contributing to the knowledge of Rhinebothrium diversity.

Intrigue surrounding the life-cycles of species of Clistobothrium (Cestoda: Phyllobothriidea) parasitising large pelagic sharks.

This study aimed to locate the adults, and thus also the definitive hosts, of three species of marine mammal-parasitising larval cestodes that have molecular affinities with Clistobothrium. New collections led to the discovery of adults of two new species of Clistobothrium, one from the longfin mako shark and one from the salmon shark. New material of Clistobothrium tumidum was collected from the great white shark and new material of a previously reported undescribed species of Clistobothrium was collected from the porbeagle shark. Larvae of Clistobothrium were opportunistically collected from sockeye salmon and four species of small squaliform sharks. Sequence data for the D1-D3 region of the 28S rDNA gene were generated for all but one of these taxa. The tree resulting from maximum likelihood analysis of those data, in combination with comparable data from GenBank, indicates that squaliform sharks can serve as intermediate hosts for the species from the porbeagle shark. The larvae from salmon exhibit a unique molecular signature and, based on diet data, may be conspecific with adults from the salmon shark. Informed by sequence data for new material of Monorygma and existing data for Phyllobothrium, the larvae provisionally identified as Monorygma grimaldii and Phyllobothrium delphini were formally transferred to Clistobothrium. Especially puzzling was that the molecular signatures of none of the eight species of Clistobothrium match those of the three marine mammal-parasitising larval forms. We are at a loss as to where else to look for the three corresponding adult forms. The great white shark remains the most likely candidate given it consumes marine mammals with some regularity, but seems unlikely to host five species of Clistobothrium. Alternatively, we are left wondering if the large marine mammal predator Carcharocles megalodon may not be extinct after all.

Redescription and taxonomic status of Dipturus chilensis (Guichenot, 1848), and description of Dipturus lamillai sp. nov. (Rajiformes: Rajidae), a new species of long-snout skate from the Falkland Islands.

Recent molecular evidence has called into question the identity of skates collected in the waters off the Falkland Islands previously identified as Zearaja chilensis. NADH2 sequence data indicate that these specimens are not conspecific with those currently referred to as Z. chilensis from Chile and, in fact, represent a novel cryptic species. This study aimed to investigate this hypothesis based on morphological comparisons of specimens from the coasts of both western and eastern South America. In total, 50 specimens from Chile and 41 specimens from the Falkland Islands were collected and examined; morphometric data were generated for a subset of specimens from both areas. NADH2 sequence data were generated for a total of 19 specimens from both areas, as well as specimens of Z. nasuta from New Zealand, D. pullopunctatus from South Africa, D. oxyrinchus from the Azores, Okamejei hollandi, and O. cairae from Borneo, and O. kenojei from Japan. Based on morphological and molecular analyses, Zearaja is synonymized with Dipturus and species assigned to the former genus are transferred to the latter genus. A neotype is designated for D. chilensis and this species is redescribed. Dipturus lamillai sp. nov. is described based on specimens from the Falkland Islands. Comparison of our NADH2 data with data for mitochondrial genomes generated from tissue samples taken from two specimens originally identified as Z. chilensis, indicate that, while the sample from Chile came from a specimen of D. chilensis, that from the skate steak obtained from a restaurant in Korea actually came from a specimen of D. lamillai sp. nov. This emphasizes the importance of confirming both the provenance and identity of specimens from which sequence data are generated and submitted to GenBank if misidentifications are to be avoided.

New insights into the identities of the elasmobranch fauna of Sri Lanka.

Identities of elasmobranchs from Sri Lanka encountered during collections conducted in an intensive nine-day survey of fish markets and landing sites at 11 localities in the North Western, Northern, and Eastern Provinces in March of 2018 were assessed. In total, 111 specimens representing 34 elasmobranch species were examined. Sequence data for the NADH2 gene were generated for all specimens. Independent Neighbor-Joining analyses, which included data for related taxa, were conducted for 25 subgroups of elasmobranchs to help confirm specific identifications. Five of the 34 species encountered are likely new to science. These consist of one species each of the batoid genera Brevitrygon, Narcine, and Torpedo, and the selachian genera Centrophorus, and Chiloscyllium. The specific identities of 12 species previously known to occur in Sri Lanka are updated to conform to current taxonomy; four of these (Gymnura cf. poecilura 2, Carcharhinus cf. limbatus, Echinorhinus sp. 1, and Iago cf. omanensis 1) represent what appear to be undescribed species reported previously from other localities. Three species (Maculabatis arabica, Acroteriobatus variegatus, and Centroscymnus owstonii) are reported from Sri Lanka for the first time; the latter species also represents the first documented record of this genus and family for the island nation. One of the two specimens on which the recent description of the new species of Planonasus indicus was based was also collected as part of this survey. Although some of the species confirmed to occur in Sri Lanka have also been found in India, others were previously known only from the Persian Gulf, Gulf of Oman, or localities in Southeast Asia. The high amount of novelty discovered as a result of a survey of such short duration emphasizes the importance of more intensive survey efforts in this region now that the civil unrest that precluded such work for nearly three decades has come to an end.

Site specificity and attachment mode of Symcallio and Calliobothrium species (Cestoda: "Tetraphyllidea") in smoothhound sharks of the genus Mustelus (Carcharhiniformes: Triakidae).

Previous studies suggest that cestodes (i.e., tapeworms) of the sister genera Symcallio and Calliobothrium attach in different specific regions of the spiral intestine of their triakid shark hosts, with species of Symcallio attaching in the anterior region of the spiral intestine and species of Calliobothrium attaching with a broader distribution centered around the middle of the spiral intestine. In the present study, we tested the generality of this pattern of site specificity in two additional species pairs: Symcallio peteri and Calliobothrium euzeti in Mustelus palumbes and S. leuckarti and C. wightmanorum in M. asterias. Finding that these cestodes also exhibit the aforementioned pattern, we investigated a series of functional explanations that might account for this phylogenetically conserved pattern of site specificity. The mucosal surface of the spiral intestine of both shark species was characterized, as were the attachment mechanisms of all four cestode species. Although anatomical differences in mucosal surface were seen along the length of the spiral intestine in both shark species, these differences do not appear to correspond to the attachment mode of these cestodes. We find that while species of Symcallio, like most cestodes, attach using their scolex, species of Calliobothrium attach with their scolex and, to a much greater extent, also with their strobila. Furthermore, attachment of Calliobothrium species appears to be enhanced by laciniations (flap-like extensions on the posterior margins of the proglottids) that interdigitate with elements of the mucosal surface of the spiral intestine. The role of proglottid laciniations in attachment in species of Calliobothrium helps reconcile a number of morphological features that differ between these two closely related cestode genera.

Three new genera of rhinebothriidean cestodes from stingrays in Southeast Asia.

Three genera of rhinebothriideans, previously referred to as New genus 1, New genus 2 and New genus 4, are erected in the the Anthocephaliidae. New genus 1 is established as Divaricobothrium gen. n., with Divaricobothrium tribelum sp. n. as its type species; Echeneibothrium trifidum Shipley et Hornell, 1906 is transferred to the genus as Divaricobothrium trifidum (Shipley et Hornell, 1906) comb. n. This genus is unique among rhinebothriidean genera in bearing bothridia that are posteriorly deeply divided into two lobes with facial loculi but no apical sucker, and a vagina that extends to near the anterior margin of the proglottid. Its species parasitise Indo-Pacific members of the genera Brevitrygon Last, Naylor et Manjaji-Matsumoto, Maculabatis Last, Naylor et Manjaji-Matsumoto and Pateobatis Last, Naylor et Manjaji-Matsumoto. New genus 2 is established as Barbeaucestus gen. n., with Barbeaucestus jockuschae sp. n. as its type species; Barbeaucestus ralickiae sp. n. is also described. Anthobothrium sexorchidum Williams, 1964 and Rhinebothrium shipleyi Southwell, 1912 are transferred to the genus as Barbeaucestus sexorchidus (Williams, 1964) comb. n. and Barbeaucestus shipleyi (Southwell, 1912) comb. n., respectively. This genus is unique among rhinebothriidean genera in that its bothridia are substantially wider than long, bear an apical sucker and at least one row of two or more facial loculi in their anterior half. Its species parasitise the genera Neotrygon Castelnau and Taeniura Müller et Henle. New genus 4 is established as Sungaicestus gen. n. with transfer of Rhinebothrium kinabatanganensis Healy, 2006, as Sungaicestus kinabatanganensis (Healy, 2006) comb. n., as its type species. Among the genera of its order, this genus most closely resembles Rhinebothrium Linton, 1890, however, despite the original description, the bothridia were found to bear, rather than lack, apical suckers. This monotypic genus is known only from the freshwater stingray Urogymnus polylepis (Müller et Henle). The familial diagnosis of the Anthocephaliidae Ruhnke, Caira et Cox, 2015 is emended. The family now houses five genera.

Redescription and Molecular Assessment of Relationships Among Three Species of Echeneibothrium (Rhinebothriidea: Echeneibothriidae) Parasitizing the Yellownose Skate, Dipturus chilensis, in Chile.

Much progress has recently been made in revising the taxonomic assignments of genera originally classified in the polyphyletic "Tetraphyllidea." Many of these genera, including Echeneibothrium, were accommodated in the order Rhinebothriidea. However, beyond this larger taxonomic action, little work has been conducted on this genus over the past 50 yr. Consequently, the criteria used for characterizing species of Echeneibothrium have lagged behind those typically used in more modern descriptions of elasmobranch-hosted cestode taxa. A series of collecting trips to Chile to obtain cestodes from the yellownose skate, Dipturus chilensis , provided a unique opportunity to apply modern morphological and molecular methods to investigate the 3 species of Echeneibothrium reported parasitizing this skate, specifically Echeneibothrium megalosoma, Echeneibothrium multiloculatum, and Echeneibothrium williamsi. In addition to redescribing all 3 species, using morphological data from light and scanning electron microscopy, maximum likelihood and bayesian inference phylogenetic analyses of the D1-D3 regions of the 28S rDNA gene were conducted to assess their relationships among other echeneibothriids for which comparable data are available. Sequencing of 59 specimens representing these 3 species of Echeneibothrium allowed us to assess the intra- and interspecific variation in the 28S rDNA gene. The redescriptions use standardized terminology for scolex morphology, proglottid anatomy, and microthrix forms and pattern; they also expand on the original descriptions to include data on scolex size, ovary size, vas deferens and vaginal configurations, testes arrangement, and genital pore position. Our morphological work led to a major reinterpretation of the scolex morphology with the recognition that all 3 species bear an apical bothridial sucker, rather than an apical loculus, prompting emendation of the diagnosis for the family Echeneibothriidae. The presence of a band of spinitriches at the apex of the apical modification of the scolex proper seems to represent an important feature for distinguishing the 2 portions of the myzorhynchus across species. Intraspecific variation ranged from 0 to 7 bp across species and interspecific variation ranged from a low of 39-46 bp between E. williamsi and E. multiloculatum to a high of 61-66 bp between E. multiloculatum and E. megalosoma. Phylogenetic analyses indicate that the 3 species of Echeneibothrium hosted by the yellownose skate are not each other's closest relatives, suggesting multiple colonization events of D. chilensis have occurred. Further phylogenetic investigation is also likely to confirm the status of the genus Pseudanthobothrium as a synonym of Echeneibothrium because its species generally group among members of Echeneibothrium.

When proglottids and scoleces conflict: phylogenetic relationships and a family-level classification of the Lecanicephalidea (Platyhelminthes: Cestoda).

This study presents the first comprehensive phylogenetic analysis of the interrelationships of the morphologically diverse elasmobranch-hosted tapeworm order Lecanicephalidea, based on molecular sequence data. With almost half of current generic diversity having been erected or resurrected within the last decade, an apparent conflict between scolex morphology and proglottid anatomy has hampered the assignment of many of these genera to families. Maximum likelihood and Bayesian analyses of two nuclear markers (D1-D3 of lsrDNA and complete ssrDNA) and two mitochondrial markers (partial rrnL and partial cox1) for 61 lecanicephalidean species representing 22 of the 25 valid genera were conducted; new sequence data were generated for 43 species and 11 genera, including three undescribed genera. The monophyly of the order was confirmed in all but the analyses based on cox1 data alone. Sesquipedalapex placed among species of Anteropora and was thus synonymized with the latter genus. Based on analyses of the concatenated dataset, eight major groups emerged which are herein formally recognised at the familial level. Existing family names (i.e., Lecanicephalidae, Polypocephalidae, Tetragonocephalidae, and Cephalobothriidae) are maintained for four of the eight clades, and new families are proposed for the remaining four groups (Aberrapecidae n. fam., Eniochobothriidae n. fam., Paraberrapecidae n. fam., and Zanobatocestidae n. fam.). The four new families and the Tetragonocephalidae are monogeneric, while the Cephalobothriidae, Lecanicephalidae and Polypocephalidae comprise seven, eight and four genera, respectively. As a result of their unusual morphologies, the three genera not included here (i.e., Corrugatocephalum, Healyum and Quadcuspibothrium) are considered incertae sedis within the order until their familial affinities can be examined in more detail. All eight families are newly circumscribed based on morphological features and a key to the families is provided. Aspects of morphological evolution and host associations are discussed in a phylogenetic context for each family and for the order as a whole. Lecanicephalidean genera lacking apical structures were confirmed as the earliest diverging lineages. Proglottid anatomy was determined to be much more conserved and indicative of phylogenetic affinities than scolex morphology. Collectively, the Lecanicephalidea parasitize three of the four orders of Batoidea-their almost exclusive absence from skates (Order Rajiformes) appears to be real; only a few records from sharks exist. At the family level, the breadth of host associations is correlated with taxonomic diversity of the family. The degree to which factors such as intermediate host use or host specificity at any stage in the life-cycle shape these patterns is currently unknown.

Pararhinebothroides-Neither the Sister-Taxon of Rhinebothroides Nor a Valid Genus.

The genus Pararhinebothroides was established for a species of cestode (Parahinebothroides hobergi) found parasitizing the Tumbes round stingray, Urobatis tumbesensis (McEachran & Chirichigno), in the inshore Pacific waters in the Gulf of Guayaquil, Ecuador. Its apparent affinities with the freshwater endemic genus Rhinebothroides were considered evidence to support the long-standing, yet controversial, biogeographical hypothesis that freshwater stingrays of the family Potamotrygonidae derived from a Pacific marine ancestor during the Cretaceous Period before the uplifting of the Andes. Here, we re-evaluate the phylogenetic and taxonomic status of P. hobergi based on examination of the available type material and newly collected material from the type host near the type locality. The new material allowed the description of tegumental structures using scanning electron microscopy and the generation of a hypothesis for the phylogenetic position of the species based on molecular data for the first time. Morphological investigations revealed that P. hobergi shares all the diagnostic features of the most recent concept of Anthocephalum, including the previously overlooked presence of bothridial apical suckers. Phylogenetic analyses based on partial 28S rDNA (D1-D3) and complete 18S rDNA sequence data for 4 specimens of P. hobergi, 45 species of other rhinebothriideans, and 5 non-rhinebothriidean outgroup species provided unequivocal support for the transfer of P. hobergi to Anthocephalum. Since this is the type and only species of the genus, Pararhinebothroides is considered a junior synonym of Anthocephalum, and Anthocephalum hobergi n. comb. is redescribed. Furthermore, our results reveal Rhinebothroides and Anthocephalum to be only distantly related among the Rhinebothriidea. Not only do our results confirm reservations expressed earlier about the affinities of P. hobergi, but they also substantially challenge inferences drawn previously about the biogeographical history of potamotrygonid stingrays based on parasitological data.

The cestode order Rhinebothriidea no longer family-less: A molecular phylogenetic investigation with erection of two new families and description of eight new species of Anthocephalum.

The spiral intestines of a total of 30 specimens of 14 species of batoids from around the world were examined for rhinebothriideans. These consisted of Taeniura grabata, Dasyatis margaritella, and Dasyatis sp. from Senegal, Dasyatis americana from Florida, Dasyatis dipterura and Dasyatis longa from México, Himantura jenkinsii, Himantura leoparda, Himantura uarnak 2, Urogymnus asperrimus 1, and Neotrygon kuhlii 4 from Australia, in addition to Himantura uarnacoides and Neotrygon kuhlii 1 from Borneo. Each of these hosted one or more species of Anthocephalum. Eleven of the cestode species were new to science; four represented described species. In addition, Urotrygon aspidura from Costa Rica hosted a species of Escherbothrium. Sufficient material was available for formal description of the following eight species of Anthocephalum: A. decrisantisorum n. sp., A. healyae n. sp., A. jensenae n. sp., A. mattisi n. sp., A. meadowsi n. sp., A. odonnellae n. sp., A. papefayi n. sp., and A. philruschi n. sp. These species differ from their nine described congeners in overall size, number of proglottids and marginal loculi, number and arrangement of testes, apical sucker size, arrangement and distribution of vitelline follicles particularly with respect in the post-poral field, and muscularity of the genital pore. The diagnosis of Anthocephalum is emended slightly to accommodate these new species. Material of four previously described Anthocephalum species, seven of the novel Anthocephalum species described here, 3 undescribed Anthocephalum species, and the species of Escherbothrium was preserved in 95% ethanol and partial 28S rDNA (D1-D3) and complete 18S rDNA sequence data were generated de novo. These data were combined with data from GenBank for Anthocephalum cf. centrurum (recognized as A. mattisi n. sp. below) and 29 species representing 12 other putative rhinebothriidean genera. Phylogenetic analyses using Bayesian Inference and Maximum Likelihood methods were conducted using a total of five representatives of the Lecanicephalidea, Cathetocephalidea and "Tetraphyllidea" as outgroups. The analyses yielded trees that were largely congruent and that supported the existence of four major subgroups of rhinebothriideans. Family designations were established for each of these clades. Echeneibothriidae was elevated from subfamily to family level to accommodate the group consisting of Echeneibothrium and Pseudanthobothrium; this family is unique in retaining the apical organ (as a myzorhynchus) into adulthood. Rhinebothriidae was elevated from subfamily to family level to accommodate the group consisting of Rhabdotobothrium, Rhinebothrium, Rhinebothroides, Rhodobothrium, Scalithrium and Spongiobothrium. This family is distinctive in its lack of apical suckers and also of a definitive anterior/posterior orientation to the bothridia. Anthocephaliidae n. fam. was established to house Anthocephalum and taxa identified as New Genus 1, New Genus 2, and New Genus 4 by previous authors. The bothridia of its members exhibit a conspicuous anterior/posterior orientation signaled by the presence of an apical sucker. In addition, its members bear marginal loculi or one or more rows of facial loculi and vitelline follicles that are usually interrupted by the ovary. Escherbothriidae n. fam. was established to house Escherbothrium and the taxon identified as New Genus 3 by previous authors. It most closely resembles Anthocephaliidae but the facial loculi are arranged in columns anteriorly and rows posteriorly, rather than arranged in multiple rows or entirely lacking. A key to the families is also provided. 

The dismantling of Calliobothrium (Cestoda: Tetraphyllidea) with erection of Symcallio n. gen. and description of two new species.

This paper aims to resolve the dual composition of the triakid shark-hosted tetraphyllidean genus Calliobothrium--an issue that has been recognized for over a decade. As it stands, this genus includes a number of large species with laciniate proglottids, most of which bear 3 suckers at the anterior margin of each bothridium, but it also includes a number of species that lack proglottid laciniations and bear only a single sucker per bothridium, most of which are relatively small. Discovery of 2 new species, 1 of each form, parasitizing the whitespot smoothhound shark, Mustelus palumbes, off South Africa, prompted the first molecular analysis of the genus. Bayesian inference and maximum likelihood analyses of 28S rDNA (D1-D3) sequence data generated for specimens of both new species as well as of 2 known species of the laciniate form (Calliobothrium australis and Calliobothrium cf. verticillatum) and 3 known species of the non-laciniate form (Calliobothrium violae, Calliobothrium riseri, and Calliobothrium barbarae) confirmed the reciprocal monophyly of the 2 clades, supporting establishment of a new genus. Because the type of Calliobothrium, C. verticillatum, is of the laciniate form, Symcallio n. gen., with Symcallio peteri n. gen., n. sp., from M. palumbes described as its type, is established to house members of the non-laciniate clade. The 11 described species consistent with this form are transferred to the new genus. A new species of the laciniate clade, Calliobothrium euzeti n. sp., is described from M. palumbes, and a revised diagnosis of Calliobothrium is presented.