Evolutionary affinities and morphological characterization of the enigmatic Zonocotyle bicaecata (Trematoda: Paramphistomoidea: Zonocotylidae) from the Upper Paraná River basin.

Neotropical fish amphistomes represent a highly diverse group within the Paramphistomoidea, with wide distribution across major South American hydrological drainages. However, the limited molecular characterization of these taxa has impeded a comprehensive assessment of their evolutionary relationships and the systematic relevance of morphological features in classification schemes. Our study, based on the critical evaluation of the type material of both nominal species of Zonocotyle (type genus of the monotypic Zonocotylidae), and newly collected specimens of Zonocotyle bicaecata from Steindachnerina insculpta (Curimatidae) in the Upper Paraná River basin, Brazil, presents a morphological reappraisal of Z. bicaecata and provides molecular data (28S rDNA, ITS1-5.8S-ITS2 region, and COI mtDNA) to assess its phylogenetic relationships. Our phylogenetic analyses confirm this species belongs to the Paramphistomoidea. The most comprehensive analyses (based on 28S and COI) further indicate a close relationship with other fish amphistomes from the Neotropical region. Additionally, we emphasized the necessity for a new classification within Paramphistomoidea and briefly discussed the host range of Zonocotyle among curimatid fish hosts.

A new cryptic species of Echinostoma (Trematoda: Echinostomatidae) closely related to Echinostoma paraensei found in Brazil.

Echinostoma paraensei, described in Brazil at the end of the 1960s and used as a biological model for a range of studies, belongs to the 'revolutum' complex of Echinostoma comprising species with 37 collar spines. However, molecular data are available only for a few isolates maintained under laboratory conditions, with molecular prospecting based on specimens originating from naturally infected hosts virtually lacking. The present study describes Echinostoma maldonadoi Valadão, Alves & Pinto n. sp., a species cryptically related to E. paraensei found in Brazil. Larval stages (cercariae, metacercariae and rediae) of the new species were found in the physid snail Stenophysa marmorata in the State of Minas Gerais, Brazil, the same geographical area where E. paraensei was originally described. Adult parasites obtained experimentally in Meriones unguiculatus were used for morphological (optical microscopy) and molecular [28S, internal transcribed spacer (ITS), nad1 and cox1] characterization. The morphology of larval and adult parasites (most notable the small-sized dorsal spines in the head collar), associated with low (0­0.1%) molecular divergence for 28S gene or ITS region, and only moderate divergence for the mitochondrial cox1 gene (3.83%), might suggest that the newly collected specimens should be assigned to E. paraensei. However, higher genetic divergence (6.16­6.39%) was found in the mitochondrial nad1, revealing that it is a genetically distinct, cryptic lineage. In the most informative phylogenetic reconstruction, based on nad1, E. maldonadoi n. sp. exhibited a strongly supported sister relationship with E. paraensei, which may indicate a very recent speciation event giving rise to these 2 species.

Size does not matter: molecular phylogeny reveals one of the largest trematodes from vertebrates, the enigmatic Ithyoclinostomum dimorphum, as a species of Clinostomum (Trematoda: Clinostomidae).

Despite the recent advances raised in the molecular era to the taxonomic knowledge of species of the family Clinostomidae, especially those belonging to the specious genus Clinostomum, some groups of these vertebrate parasites remain poorly studied. This is the case of species of the enigmatic genus Ithyoclinostomum Witenberg, 1926, until recently monotypic and restricted to South America, but with its occurrence expanded to North America after the description of I. yamagutii Rosser et al., 2020. Nevertheless, molecular data for the type species of the genus, Ithyoclinostomum dimorphum (Diesing, 1850), is lacking so far. In the present study, large clinostomid metacercariae morphologically indistinguishable from I. dimorphum were obtained from two erythrinid fishes from the Rio Doce River, Southeast Brazil. Samples of the parasites were subjected to a multigene (28S rDNA, ITS and cox1) molecular characterization followed by phylogenetic reconstructions. Phylogenies based on single-gene and concatenated datasets revealed unequivocally that I. dimorphum falls in a well-supported clade together with species of the genus Clinostomum. Moreover, the molecular divergences observed in relation to Clinostomum spp. [ranges of 2.4-6%, 2.4-3.8% and 14.7-19.3% for the ITS, 28S and cox1 genes, respectively] are compatible with a congeneric status with these species. Therefore, the genus Ithyoclinostomum is here synonymized with Clinostomum Leidy, 1856 and C. dimorphum (Diesing, 1850) Braun, 1899 re-established. In the phylogenetic analysis, the recently described 'Ithyoclinostomum' yamagutii, presented as an isolated, independent lineage, showing significant molecular divergences to C. dimorphum (12.6%, 7.6%, 18,6% for the ITS, 28S and cox1 genes, respectively). However, given the complex scenario raised in the morphology-based taxonomy of Clinostomidae, we took a conservative approach by not proposing a new genus to 'I.' yamagutii until molecular data of other clinostomid genus from birds, Clinostomatopsis, become available. Data here presented reveals that body size is not a useful criterion for higher-level classification in Clinostomidae. Finally, we highlighted the importance of the availability of molecular data for the type species of trematode genera proposed from South America to support a trans- or intercontinental distribution.

A new species of Echinostoma (Trematoda: Echinostomatidae) from the 'revolutum' group found in Brazil: refuting the occurrence of Echinostoma miyagawai (=E. robustum) in the Americas.

Although Echinostoma robustum (currently a synonym of E. miyagawai) was reported in the Americas based on molecular data, morphological support on adult parasites is still required. Herein, a new species of Echinostoma is described based on worms found in a chicken from Brazil. Molecular phylogenetic analyses based on 28S (1063 bp), ITS (947 bp) and Nad-1 (442 bp) datasets reveal the inclusion of the new species within Echinostoma 'revolutum' species complex. Moreover, it was verified the conspecificity between cercariae previously identified as E. robustum in Brazil [identical ITS and only 0.3% of divergence (1 nucleotide) in Nad-1]. Species discovery analyses show that these two isolates form an independent lineage (species) among Echinostoma spp. Compared to E. miyagawai, the new species presents relatively high divergence in Nad-1 (7.88­9.09%). Morphologically, the specimens are distinguished from all nominal species from the 'revolutum' species complex by the more posterior position of the testes (length of post-testicular field as a proportion of body length about 20%). They further differ from E. miyagawai and South American Echinostoma spp. by the higher proportion of forebody to the body length. Therefore, combined molecular and morphological evidence supports the proposal of the species named here as Echinostoma pseudorobustum sp. nov.

NEW RECORDS OF NEMATODES IN THE YELLOW-BILLED CUCKOO COCCYZUS AMERICANUS (CUCULIFORMES: CUCULIDAE) BASED ON A MIGRANT SPECIMEN FOUND IN BRAZIL.

The yellow-billed cuckoo, Coccyzus americanus, is a threatened passerine bird native to North America that migrates to overwinter in South America. Although migratory birds have drawn broad attention, given their assumed role in the long-distance dispersal of parasites, studies on the helminth fauna of this cuculid are scarce. In the present study, nematodes found in the gastrointestinal tract of a specimen of C. americanus found in Belo Horizonte, State of Minas Gerais, southeastern Brazil, were characterized morphologically. Five species of gastrointestinal nematodes belonging to 4 families were identified: Synhimantus (Dispharynx) nasuta (Rudolphi, 1819) and Synhimantus (Dispharynx) resticulaCanavan, 1929 (Acuariidae), Microtetrameres sp. (Tetrameridae), and Cyrnea piayaeSandground, 1929 (Habronematidae) and Subulura halli Barreto, 1918 (Subuluridae). Except for Microtetrameres sp., all other nematodes are reported in this host for the first time. Although it is difficult to accurately determine the geographical origin of infections, it is important to note that Sy. (D.) resticula and Su. halli are reported for the first time in Brazil. Additionally, the absence of the former species infecting hosts where the widespread and generalist Sy. (D.) nasuta was found may indicate that Sy. (D.) resticula have either been overlooked in previous studies or that this nematode is rare indeed. Finally, the geographical distribution of Su. halli is greatly expanded and now includes the Americas. Aspects related to the dispersion of these parasites, both in migrating and native hosts, are briefly discussed.

Ophiotaenia echidis n. sp. (Cestoda: Proteocephalidae) from the saw-scaled viper, Echis carinatus sochureki Stemmler (Ophidia: Viperidae), one of the world's deadliest snakes, from the United Arab Emirates.

Ophiotaenia echidis n. sp. (Cestoda: Proteocephalidae) is described from the intestine of one of the world's deadliest snakes, the saw-scaled viper Echis carinatus sochureki Stemmler (Ophidia: Viperidae) in the United Arab Emirates. The new species differs from other species of the non-monophyletic Ophiotaenia by the position of testes in two longitudinal lines on both sides of the uterus, and by the large size of an embryophore (diameter of 44-55 µm versus less than 40 µm in other species). Phylogenetic reconstructions based on lsrDNA and concatenated lsrDNA + COI datasets place the new species among proteocephalids from unrelated zoogeographical realms but mostly infecting venomous snakes. In all analyses, O. echidis n. sp. exhibited a strongly supported sister relationship with O. lapata Rambeloson, Ranaivoson et de Chambrier, 2012, a parasite of a pseudoxyrhophiid snake endemic to Madagascar. Despite a shared close evolutionary history between these taxa, morphological synapomorphies remain unclear, which impedes the erection of a new genus to accommodate them. A list of the 71 tapeworms of the former, non-monophyletic subfamily Proteocephalinae, parasitising snakes and lizards, including species inquirendae, and the phylogenetically closely related Thaumasioscolex didelphidis from opossum, with selected characteristics, is also provided, together with a checklist of helminth parasites reported from E. carinatus.

New arrangement of three genera of fish tapeworms (Cestoda: Proteocephalidae) in catfishes (Siluriformes) from the Neotropical Region: taxonomic implications of molecular phylogenetic analyses.

Tapeworms (Cestoda: Proteocephalidae) are the dominant component of communities of intestinal parasites in pimelodid and other catfishes (Siluriformes) from South America. Even though these parasites have been studied intensively over more than one century, molecular taxonomy and phylogenetics have questioned their morphology-based classification, thus raising doubts about the systematic value of traits commonly used to circumscribe individual taxa. In the present study, members of three morphologically well-characterized genera of proteocephalids from pimelodid (Hemisorubim platyrhynchos and Sorubim lima) and auchenipterid (Ageneiosus inermis) catfishes from the Paraná or Amazon River basins were subjected to DNA sequencing of the large subunit nuclear ribosomal RNA (lsrDNA) and complete mitochondrial cytochrome c oxidase subunit I (COI). Phylogenetic analyses revealed the sister relationship between Manaosia bracodemoca and Mariauxiella piscatorum, and among Mariauxiella pimelodi and Ageneiella brevifilis. As a result, Mar. piscatorum and A. brevifilis are transferred to Manaosia and Mariauxiella, respectively, as Manaosia piscatorum n. comb. and Mariauxiella brevifilis n. comb., and the genus Ageneiella is suppressed. Diagnoses of Manaosia and Mariauxiella are amended. In addition, the present study revealed misidentification of tapeworms whose sequences are deposited in the GenBank database.

Exploring Neotropical anuran parasites: a morphological, life cycle and phylogenetic study of Catadiscus marinholutzi (Trematoda: Diplodiscidae).

Amphistome species belonging to the genus Catadiscus are poorly studied intestinal trematodes found primarily in Neotropical anurans. Herein, developmental stages of an amphistome species found during herpetological and malacological surveys in a temporary marsh pond from Brazil were subjected to morphological (light and scanning electron microscopy) and molecular analyses. Adult parasites recovered from anurans were identified as Catadiscus marinholutzi. Amphistome cercariae found in the planorbid snails Drepanotrema depressissimum and Drepanotrema lucidum from the same waterbody were used for experimental and molecular studies. Immature parasites, morphologically compatible with members of Catadiscus, were experimentally obtained in laboratory-reared tadpoles. Sequencing of a partial region of 28S rDNA gene of both adult and cercariae revealed 100% similarity between these developmental stages, confirming their conspecificity. Phylogenetic analyses were attempted for the first time to reveal the position of a species of Catadiscus in the superfamily Paramphistomoidea. Catadiscus marinholutzi falls in a virtual polytomy together with other paramphistomoids, which leaves its phylogenetic relationships within the group unclear. Moreover, the high genetic divergence to Diplodiscus spp. (10.06­10.84%) cast doubts on the placement of Catadiscus within Diplodiscidae. Hence the species composition of the Diplodiscidae should be re-evaluated in further studies using a broader spectrum of related taxa.

A phylogenetic study of the cecal amphistome Zygocotyle lunata (Trematoda: Zygocotylidae), with notes on the molecular systematics of Paramphistomoidea.

Zygocotyle lunata inhabits the caecum of birds and mammals from the American continent. This amphistome parasite is easily maintained in the laboratory and serves as a model organism in life-cycle studies, but it has seldom been studied using molecular data. Neither the position of Z. lunata in the superfamily Paramphistomoidea nor the monophyly of the Zygocotylidae has been evaluated with molecular phylogenetic methods. In the present study, adult specimens of Z. lunata obtained experimentally in mice from Brazil were submitted to molecular studies. Partial sequences of nuclear (1261 bp of 28S and 418 bp of 5.8S-ITS-2) and mitochondrial (1410 bp of cytochrome c oxidase 1, cox1) markers were compared with published data. In the most well-resolved phylogeny, based on 28S sequences, Z. lunata clustered in a well-supported clade with Wardius zibethicus, the only other species currently included in the Zygocotylidae, thus confirming the validity of this family. Divergence of 28S sequences between these species was 2.2%, which falls in the range of intergeneric variation (0.9-5.6%) observed in the other two monophyletic groups in the 28S tree, i.e., representatives of Gastrodicidae and Neotropical cladorchiids (Cladorchiidae). Analysis of ITS-2 and two parts of the cox1 gene placed Z. lunata within poorly resolved clades or large polytomies composed of several paramphistomoid families, without clarifying higher-level phylogenetic relationships. The cox1 of a Brazilian isolate of Z. lunata is 99.6% similar to a Canadian isolate, confirming the pan-American distribution of the species. Finally, our phylogenetic reconstructions of Paramphistomoidea revealed a complex scenario in the taxonomic composition of some amphistome families, which highlights a need for further integrative studies that will likely result in rearrangements of traditional morphology-based classifications.

Molecular data support monophyly of the recently erected Riggenbachiella (Cestoda: Proteocephalidae), parasites of Neotropical catfishes.

Proteocephalid tapeworms (Cestoda) are one of the dominant groups of intestinal parasites of Neotropical catfishes (Siluriformes), especially pimelodids (de Chambrier et al. 2015). The long and convoluted taxonomic history of species of Chambriella Rego, Chubb Pavanelli, 1999 has been disentangled only recently by Alves et al. (2017). These authors erected a new genus, Riggenbachiella Alves, de Chambrier, Luque Scholz, 2017, to accommodate R. amazonense Alves, de Chambrier, Luque Scholz, 2017 (type-species) and R. paranaense (Pavanelli Rego, 1989) (previously Chambriella paranaensis), parasites of pimelodid catfishes.

Towards a robust systematic baseline of Neotropical fish tapeworms (Cestoda: Proteocephalidae): amended diagnoses of two genera from the redtail catfish, Phractocephalus hemioliopterus.

Species of two monotypic genera, Ephedrocephalus Diesing, 1850 and Zygobothrium Diesing, 1850 (Cestoda: Proteocephalidae), parasites of one of the most basal members of the catfish family Pimelodidae, the redtail catfish, Phractocephalus hemioliopterus, in the Neotropical Region, are redescribed based on the evaluation of type specimens and newly collected material. Generic diagnoses are amended to provide a robust baseline for the future re-arrangement of the classification of proteocephalid cestodes. Ephedrocephalus is typified by the medullary position of the ovary and uterus, the cortical distribution of vitelline follicles (dispersed throughout almost the entire ventral cortex) and the testes in one dorsal field. Zygobothrium is primarily characterized by its possession of a robust scolex bearing four uniloculate suckers with two openings each and by the tetralobed velum (laciniations) on every proglottid (two on the ventral and two on the dorsal side). The redtail catfish is the definitive host of as many as seven species of proteocephalid cestodes, which, however, do not represent a monophyletic group. Some species, including Z. megacephalum, are among the earliest diverged parasites of Neotropical catfishes, being closely related to African and North American proteocephalids from catfishes, whereas others such as E. microcephalus belong to more recently diverged taxa with uncertain interrelations. Unlike most proteocephalids of the redtail catfish, which almost always infect the anterior parts of the host intestine, E. microcephalus and Z. megacephalum occur exclusively in its posterior third. A key to the identification of the proteocephalid species parasitizing P. hemioliopterus is also presented.

Annotated checklist of fish cestodes from South America.

An exhaustive literature search supplemented by a critical examination of records made it possible to present an annotated checklist of tapeworms (Cestoda) that, as adults or larvae (metacestodes), parasitize freshwater, brackish water and marine fishes, i.e. cartilaginous and bony fishes, in South America. The current knowledge of their species diversity, host associations and geographical distribution is reviewed. Taxonomic problems are discussed based on a critical evaluation of the literature and information on DNA sequences of individual taxa is provided to facilitate future taxonomic and phylogenetic studies. As expected, the current knowledge is quite uneven regarding the number of taxa and host-associations reported from the principal river basins and marine ecoregions. These differences may not only reflect the actual cestode richness but may also be due to the research effort that has been devoted to unravelling the diversity of these endoparasitic helminths in individual countries. A total of 297 valid species, 61 taxa identified to the generic level, in addition to unidentified cestodes, were recorded from 401 species of fish hosts. Among the recognized cestode orders, 13 have been recorded in South America, with the Onchoproteocephalidea displaying the highest species richness, representing c. 50% of all species diversity. The majority of records include teleost fish hosts (79%) that harbour larval and adult stages of cestodes, whereas stingrays (Myliobatiformes) exhibit the highest proportion of records (39%) among the elasmobranch hosts. Fish cestodes are ubiquitous in South America, being mostly recorded from the Warm Temperate Southeastern Pacific (WTSP; 31%) for marine hosts and the Amazon River basin (45%) for freshwater ones. The following problems were detected during the compilation of literary data: (i) unreliability of many records; (ii) poor taxonomic resolution, i.e. identification made only to the genus or even family level; (iii) doubtful host identification; and (iv) the absence of voucher specimens that would enable us to verify identification. It is thus strongly recommended to always deposit representative specimens in any type of studies, including faunal surveys and ecological studies. An analysis of the proportion of three basic types of studies, i.e. surveys, taxonomic and ecological papers, has shown a considerable increase of ecological studies over the last decade.

A checklist of the aspidogastrea (platyhelminthes: trematoda) of the world.

A checklist of records of aspidogastrean trematodes (Aspidogastrea) is provided on the basis of a comprehensive survey of the literature since 1826, when the first aspidogastrean species was reported, until December 2014. We list 61 species representing 13 genera within 4 families and 2 orders of aspidogastreans associated with 298 species of invertebrate and vertebrate hosts. The majority of records include bivalves (44% of the total number of host-parasite associations), whereas records from bony fishes represent 32% of host-parasite associations. The aspidogastreans have worldwide distribution, with the highest number of records in the Neartic Region for freshwater hosts and the North Atlantic Ocean for marine ones. The checklist includes a parasite-host list with data on host habitat, site of infection and distribution area of parasites, and a host-parasite list. A limited number of molecular studies on aspidogastreans does not allow us to unravel phylogenetic relationships within the Aspidogastrea.

Physaloptera bainae n. sp. (Nematoda: Physalopteridae) parasitic in Salvator merianae (Squamata: Teiidae), with a key to Physaloptera species parasitizing reptiles from Brazil.

Physaloptera bainae n. sp. (Nematoda: Physalopteridae) from stomach of the large "tegú" lizard Salvator merianae (Squamata: Teiidae), collected in an ecological park that is part of the Atlantic Rainforest biome in Juiz de Fora, Minas Gerais, Brazil, is described. The new species which has females with didelphic uterus, is the only species of the genus exhibiting external teeth in the form of 4 spines that are organized in a cross-shaped pattern at the anterior apical end, with 2 minute adcloacal papillae on the anterior part of cloacal aperture in males and a large cuticular expansion at the anterior end of females. Moreover, P. bainae n. sp. can be differentiated from the other congeners by the combination of other features, e.g., number (23) and pattern of caudal papillae, spicule size (left 554-600; right 589-617) and vulval position (on the first third of body), and because the specimens are particularly large and robust. Physaloptera monodens is considered a junior synonym of P. obtusissima. In addition, a key to Physaloptera species parasitizing reptiles from Brazil is provided.

A new Physaloptera (Nematoda: Physalopteridae) parasite of Tupinambis merianae (Squamata: Teiidae) from southeastern Brazil.

Physaloptera tupinambae n. sp. (Nematoda: Physalopteridae) collected from the stomach of 1 Tupinambis merianae (Squamata: Teiidae) male lizard is described. Physaloptera tupinambae, which belongs to the didelphys group, is the only species in the genus that exhibits a bipartite internal tooth and in which the excretory pore is anterior to the deirids. The new species can be differentiated from the other congeners mainly based on the number and pattern of caudal papillae and the length and shape of spicules. Physaloptera murisbrasiliensis and Physaloptera clausa orientalis are the only physalopterids with the same number of caudal papillae as for P. tupinambae, but they differ from the new species mainly in length and shape of spicules. Based on morphological analysis, the speciation process among Physaloptera probably occurred via host capture, and P. tupinambae occupies an intermediate phylogenetic position in the genus. Most likely, the new parasite was acquired by the lizard through ingestion of an intermediate host. Finally, Physaloptera calotisi, Physaloptera funambuli, Physaloptera guptae, Physaloptera indica, Physaloptera johnsoni, Physaloptera kherai, and Physaloptera thaparus are moved to Abbreviata.