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The first molecular assessment of phylogenetic relationships of cladorchiid digeneans (superfamily Paramphistomoidea Fischoeder, 1901) from freshwater fishes based on 28S rDNA, ITS2 and cox1 sequences reveals the subfamilies Dadayiinae Fukui, 1929 and Kalitrematinae Travassos, 1933 as non-monophyletic, whereas Dadaytrema Travassos, 1931 represented by three species is monophyletic. Fourteen species of cladorchiids were found in characiform, perciform and siluriform fishes in the Neotropical Region (Brazil and Peru), with numerous new host and geographical records. The first scanning electron micrographs of seven species are presented. Two new species of dadayiine and one new species of kalitrematine paramphistomes are described. Microrchis macrovarium sp. n. from Pimelodella cristata (Müller et Troschel) (type host), Tetranematichthys quadrifilis (Kner) and Pterodoras granulosus (Valenciennes) in Brazil and Peru differs from all three congeners in that the testes are directly tandem, not oblique, and that the ovary, between the caecal ends, is widely separated from the testes. Pronamphistoma philippei sp. n. from Heros sp. in Brazil is distinguished from the type and only species, Pronamphistoma cichlasomae Thatcher, 1992, by the absence of the anterior collar-like expansion present in the type species, the presence of extramural rather than intramural pharyngeal sacs, and the unusual development of the dorsal and ventral exterior circular muscle fibre series in the acetabulum. Pseudocladorchis romani sp. n. from P. granulosus (type host), Brachyplatystoma vaillantii (Valenciennes), Calophysus macropterus (Lichtenstein), Megalodoras uranoscopus (Eigenmann et Eigenmann) and Oxydoras niger (Valenciennes) in Brazil and Peru, is most similar to Pseudocladorchis nephrodorchis Daday, 1907 but differs in the shape of the testes (irregular, versus reniform in the latter species) and the size of the ovary (as large as, or larger than, the testes in the new species). The generic diagnosis of Pronamphistoma Thatcher, 1992 is amended. Dadaytremoides parauchenipteri Lunaschi, 1989 is transferred to Doradamphistoma Thatcher, 1979 as D. parauchenipteri (Lunaschi, 1989) comb. n. based on morphological and molecular evidence.

Despite recent advances in the molecular knowledge of amphistome trematodes, most genera known from fish remain to be genetically characterized. This is the case for Kalitrema, a genus of the speciose family Cladorchiidae and the type of Kalitrematinae. The type and only species of this genus, Kalitrema kalitrema Travassos, 1933, was originally proposed based on two specimens found in an armored suckermouth catfish from Brazil, and its phylogenetic position has not been evaluated. In this study, paramphistomes found in Hypostomus alatus (2/9; 22.2%) and Hypostomus francisci (4/143; 2.8%) from the Paraobepa River (São Francisco River basin), Minas Gerais, Brazil, between December 2019 and November 2021, were subjected to morphological study. The parasites were identified in low intensity of infection [1.2 (1–2)] and redescribed as K. kalitrema. This species exhibits unique features such as a linguiform body with a circular ridge near the anterior end and a deep, median notch present at the posterior extremity of the body, apparently dividing the body into two lobes. A subset of specimens was further subject to phylogenetic analyses based on the most densely sampled markers, the nuclear ribosomal RNA (28S and ITS2) and mitochondrial cox1, which revealed the inclusion of K. kalitrema in a Neotropical clade of fish paramphistomes. The most comprehensive phylogenetic tree, based on the 28S dataset, confirmed K. kalitrema as an independent, early diverging lineage among Neotropical fish cladorchiids. However, the monophyly of Kalitrematinae was not sustained, given that species of the other kalitrematine genera Pseudocladorchis and Iquitostrema included in the phylogenetic analysis fell in a distinct clade with other fish cladorchiids. As a result, we propose here a narrower concept for Kalitrematinae sensu stricto, accommodating only Kalitrema (type genus) until a more natural subfamilial or familial classification is provided. [ABSTRACT FROM AUTHOR]