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a new species of deep-sea digenean, parasitizing the gallbladder of the \"Bigeye grenadier\" ( Günther, 1878) in the deep waters of the southeastern Pacific Ocean is described on the basis of morphological and molecular (28S rRNA) data. The new species is distinguishable from Yamaguti, 1940, the only other member of the genus, by its subterminal oral sucker, the position of the ovary and testes, the larger anterior seminal vesicle compared to the posterior one, and its larger eggs. In addition, the new species is a parasite of a deep-sea fish, whereas is a parasite of shallow-water fish. A phylogenetic tree, based on 28S rDNA sequences, indicates that this species is included in a clade of deep-sea fellodistomid species ( spp.). We provide the first molecular data on the genus Yamaguti, 1940 and expand the molecular database for the family Fellodistomidae. Further studies, including sequences from other fellodistomid taxa, are needed to more precisely infer relationships within this family.

Trematodes of the family Allocreadiidae are primarily found in the intestines of freshwater fishes around the world. The family includes 15 genera and c. 130 species. The last 2 decades have witnessed an increase in the genetic library of its species. Molecular data have been crucial for species delimitation and species description within Allocreadiidae and for understanding their evolutionary and biogeographical history and classification. Here, the mitogenomes of 3 species of allocreadiids were obtained using high throughput sequencing methods. Mitogenomes were compared with other members of the order Plagiorchiida to determine their molecular composition, gene rearrangement and phylogenetic interrelationships. The complete circular mitogenomes of Allocreadium lobatum, Creptotrematina aguirrepequenoi and Wallinia mexicana were 14 424, 13 769 and 13 924 bp long respectively, comprising 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions. Gene arrangements were identical to other Xiphidiatan trematodes. Phylogenetic analyses using the mitogenomes revealed Allocreadiidae as a monophyletic group closely related to other members of the suborder Xiphidiata; A. lobatum was yielded as the sister taxon of C. aguirrepequenoi + W. mexicana. Our study increases the complete mitochondrial genome library of trematodes and strengthens our understanding of the phylogenetic relationships and classification of this parasite group.

Background The Bucephalidae is a large family of digenean trematodes but most previous analyses of its phylogenetic position have relied on a single mitochondrial gene or morphological features. Mitochondrial genomes (mitogenomes) remain unavailable for the entire family. To address this, we sequenced the complete mitogenome of Dollfustrema vaneyi and analyzed the phylogenetic relationships with other trematodes. Results The circular genome of Dollfustrema vaneyi spanned 14,959 bp and contained 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a major non-coding region. We used concatenated amino acid and nucleotide sequences of all 36 genes for phylogenetic analyses, conducted using MrBayes, IQ-TREE and PhyloBayes. We identified pronounced topological instability across different analyses. The addition of recently sequenced two mitogenomes for the Aspidogastrea subclass along with the use of a site-heterogeneous model stabilized the topology, particularly the positions of Azygiidae and Bucephalidae. The stabilized results indicated that Azygiidae was the closest lineage to Bucephalidae in the available dataset, and together, they clustered at the base of the Plagiorchiida. Conclusions Our study provides the first comprehensive description and annotation of the mitochondrial genome for the Bucephalidae family. The results indicate a close phylogenetic relationship between Azygiidae and Bucephalidae, and reveal their basal placement within the order Plagiorchiida. Furthermore, the inclusion of Aspidogastrea mitogenomes and the site-heterogeneous model significantly improved the topological stability. These data will provide key molecular resources for future taxonomic and phylogenetic studies of the family Bucephalidae.

Opegaster Ozaki, 1928 is one of the most species-rich genera of the trematode family Opecoelidae, commonly found in marine teleost fishes. This study describes a new species of Opegaster of the intertidal fish species Labrisomus xanti Gill (Labrisomidae) (type host) and Tomicodon zebra (Jordan et Gilbert) (Gobiesocidae) from the coast of Oaxaca, Mexican Pacific. Opegaster mezcal sp. n. is characterised by the absence of papillae on the ventral sucker and the size and shape of the testes. Sequences of the D1-D3 domains from large subunit from nuclear DNA (28S) were generated for the new species and for another opecoelid species, Opecoelus adsphaericus Manter et Van Cleave, 1951. The new sequences of Opegaster mezcal sp. n. formed a clade, which is sister to O. adsphaericus. However, the phylogenetic relationships among genera of Opecoelinae remain unresolved because few species have been analysed; the addition of further taxa is necessary to better understand the evolution of the subfamily.

Prohemistomum vivax is a zoonotic small cyathocotylid trematode that inhabits the intestines of fish-eating birds and mammals. Here, we amplified the internal transcribed spacer (ITS) sequence and six mitochondrial protein-coding genes (PCGs) from P. vivax . The ITS region was 1389 base pairs long and had a partial 18S ribosomal RNA gene, a full ITS1, 5.8S rRNA, and ITS2 sequence, and a partial 28S rRNA gene. The ITS region of P. vivax showed a minimum pairwise distance (0.3–0.6%) from the ITS sequences of Cyathocotylidae sp. 1 and 2 metacercariae from Clarias gariepinus . This result suggests that these metacercariae belong to P. vivax metacercariae. We first amplified mitochondrial genes from P. vivax , including cytochrome c oxidase subunit III ( cox 3) partial sequence; tRNA-His, cytochrome b ( cyt b), and NADH dehydrogenase subunit 4L ( nad 4L) complete sequences; and NADH dehydrogenase subunit 4 ( nad 4), cytochrome c oxidase I ( cox 1), and NADH dehydrogenase subunit 5 ( nad 5) partial sequences. P. vivax was most closely related to Cyathocotyle prussica (NC_039780) and Holostephanus sp. (OP082179), with cox 1, cox 3, and cyt b genes conserved among the three trematodes. The ML phylogenetic tree of ITS sequences supports the order Diplostomida, divided into two main clades (the superfamily Diplostomoidea and Schistosomatoidea). The phylogeny of concatenated amino acid sequences of P. vivax six PCGs revealed that diplostomoids and Clinostomum sp. evolved in a clade with Plagiorchiida members, away from Schistosoma species. These results may yield ribosomal and mitochondrial genetic markers for molecular epidemiological investigations of cyathocotylid intestinal flukes.

Background The Eucotylidae Cohn, 1904 (Superfamily: Microphalloidea), is a family of digeneans parasitic in kidneys of birds as adults. The group is characterized by the high level of morphological similarities among genera and unclear systematic value of morphological characters traditionally used for their differentiation. In the present study, we sequenced the complete or nearly complete mitogenomes (mt genome) of two eucotylids representing the genera Tamerlania ( T. zarudnyi ) and Tanaisia ( Tanaisia sp.). They represent the first sequenced mt genomes of any member of the superfamily Microphalloidea. Methods A comparative mitogenomic analysis of the two newly sequenced eucotylids was conducted for the investigation of mitochondrial gene arrangement, contents and genetic distance. Phylogenetic position of the family Eucotylidae within the order Plagiorchiida was examined using nucleotide sequences of mitochondrial protein-coding genes (PCGs) plus RNAs using maximum likelihood (ML) and Bayesian inference (BI) methods. BI phylogeny based on concatenated amino acids sequences of PCGs was also conducted to determine possible effects of silent mutations. Results The complete mt genome of T. zarudnyi was 16,188 bp and the nearly complete mt genome of Tanaisia sp. was 13,953 bp in length. A long string of additional amino acids (about 123 aa) at the 5′ end of the cox 1 gene in both studied eucotylid mt genomes has resulted in the cox 1 gene of eucotylids being longer than in all previously sequenced digeneans. The rrnL gene was also longer than previously reported in any digenean mitogenome sequenced so far. The TΨC and DHU loops of the tRNAs varied greatly between the two eucotylids while the anticodon loop was highly conserved. Phylogenetic analyses based on mtDNA nucleotide and amino acids sequences (as a separate set) positioned eucotylids as a sister group to all remaining members of the order Plagiorchiida. Both ML and BI phylogenies revealed the paraphyletic nature of the superfamily Gorgoderoidea and the suborder Xiphidiata. Conclusions The average sequence identity, combined nucleotide diversity and Kimura-2 parameter distances between the two eucotylid mitogenomes demonstrated that atp 6, nad 5, nad 4L and nad 6 genes are better markers than the traditionally used cox 1 or nad 1 for the species differentiation and population-level studies of eucotylids because of their higher variability. The position of the Dicrocoeliidae and Eucotylidae outside the clade uniting other xiphidiatan trematodes strengthened the argument for the need for re-evaluation of the taxonomic content of the Xiphidiata. Graphical Abstract

Two cases of human philophthalmosis have been reported in Japan. Gravid flukes removed from the eyes of the patients were broken, but their morphological characteristics suggest that an unknown species of the genus Philophthalmus is involved as a pathogen for humans. The mitochondrial DNA barcode of the human eye fluke enabled us to discover its larval stage from the Japanese mud snail, Batillaria attramentaria. The discovered cercaria had previously been temporarily described as “Philophthalmid sp. I.” In this study, we examined the infection status of B. attramentaria with Philophthalmid sp. I found on a muddy seashore of the Seto Inland Sea, Japan, and the resulting metacercariae were experimentally administered to Japanese quails to develop them into the gravid adult stage. The complete specimens of the adult and larval stages allowed us to describe a new species. Based on morphological and molecular analyses, Philophthalmus hechingeri n. sp. is proposed for the human-infecting eye fluke in Japan. The natural definitive hosts of the new species are unknown. However, the habitat of B. attramentaria suggests that shorebirds (seagulls, sandpipers, and plovers) might be the possible candidates.

Cyclocoelidae Stossich, 1902 are medium-sized to large digenean bird parasites. Although these parasites bear few visible autapomorphic signs, and their diagnostic characters are unstable in response to the pressure applied during preparation, the numerous hitherto suggested re-classifications within the family have not been supported by any molecular analysis. We analyse here cyclocoelids found during the extensive examination of central European birds performed from 1962 to 2016, provide comparative measurements, host spectra, prevalence and intensity, and provide and analyse sequences of four DNA loci of five of the cyclocoelid species. Cyclocoleum Brandes, 1892 appears paraphyletic; thus we suggest the re-classification of Cyclocoleum obscurum (Leidy, 1887) as Harrahium obscurum (Leidy, 1887) Sitko and Heneberg comb. n. Molecular phylogenetics questioned also the validity of Cyclocoelinae Stossich, 1902 and Hyptiasminae Dollfus, 1948, which formed a single clade, whereas Allopyge Johnston, 1913, Prohyptiasmus Witenberg, 1923 and Morishitium Witenberg, 1928 formed another clade. Haematotrephinae Dollfus, 1948 are newly characterized as having a pretesticular or intertesticular ovary that forms a triangle with the testes. Analyses of non-European genera of the Cyclocoelidae and an examination of the position of families within Echinostomata La Rue, 1926 are needed.

The genus Maritrema Nicoll, 1907 (Platyhelminthes, Trematoda, Plagiorchiida, Microphallidae) comprises cosmopolitan species that predominantly parasitize birds. Although approximately 65 species have been described worldwide, including 6 for Argentina, molecular data referring to Maritrema species are still scarce worldwide, especially in South America. Unfortunately, this lack of references for nucleotide sequences is an obstacle to understanding the taxonomy and life cycles of trematodes, and impedes advancing our studies on the phylogeny and geographical distribution of these parasites. For that reason, we performed the molecular study of developmental stages of Maritrema bonaerense : cercariae (collected from the snail first intermediate host Heleobia australis , inhabiting Mar Chiquita lagoon) and metacercariae (collected from the crab second intermediate host Neohelice granulata , inhabiting Mar Chiquita lagoon and San Antonio Oeste, Argentina). The accordance between the ITS2 sequence of M. bonaerense cercaria from the snail H. australis and the sequences of metacercariae from the crab N. granulata was 100%, supporting previous findings of the life cycle of M. bonaerense based on morphological data. All Maritrema species are included in a monophyletic and well-supported clade. Maritrema bonaerense grouped more closely with Maritrema gratiosum . These findings contribute to the knowledge of digeneans in coastal marine ecosystems.

The systematic position and phylogenetic relationships of the family Cortrematidae Yamaguti, 1958 have always been controversial. In the present study, the phylogenetic relationships of this family and its constituent genera and families within the superfamily Microphalloidea were evaluated using previously published and newly obtained sequences of 28S rDNA of Cortrema magnicaudata (Bykhovskaya-Pavlovskaya, 1950) (Cortrematidae), Phaneropsolus praomydis Baer, 1971 and Microtrema barusi Sitko, 2013 (Phaneropsolidae). Results clearly demonstrate that the genus Cortrema Tang, 1951 is closest to Gyrabascus Macy 1935, both genera forming one of the clades within the family Pleurogenidae in the superfamily Microphalloidea and sharing several important morphological features. Thus, the family Cortrematidae should be considered among synonyms of the Pleurogenidae. Based on the analysis of morphology, C. corti Tang, 1951, C. testilobata (Bykhovskaya-Pavlovskaya, 1953) and C. niloticus Ashour, Ahmed et Lewis, 1994 are considered junior synonyms of C. magnicaudata. The phylogenetic position of P. praomydis as a family-level branch not showing close relationships with other families of the Microphalloidea, supports the status of the Phaneropsolidae as an independent family. The genus Parabascus Looss, 1907 previously considered within the Phaneropsolidae clearly belongs to the Pleurogenidae. In addition, the molecular phylogeny has demonstrated that the recently described phaneropsolid Microtrema barusi belongs to the microphallid genus Microphallus Ward, 1901. Therefore, Microtrema Sitko, 2013 is considered a junior synonym of Microphallus. Our analysis has also confirmed the status of Collyriclidae as a family within the Microphalloidea. Not yet sequenced representatives of other families within the Microphalloidea (e.g. Anenterotrematidae, Eumegacetidae, Renschtrematidae, Stomylotrematidae, etc.) need to be included in future molecular phylogenetic studies to better unravel the taxonomic structure and content of this diverse digenean superfamily.