Multilocus, morphological, and ecological data reveal the presence of three new species of the genus Neodiplostomum Railliet 1919 (Digenea: Diplostomidae), endoparasites of birds from the Neotropical region of Mexico.

Neodiplostomum Railliet, 1919, is a cosmopolitan genus that parasitises mainly the intestines of birds of prey, and occasionally those of passerines, cuculiforms, and mammals. Adults of three undescribed species of Neodiplostomum were obtained from five bird species from three families (Accipitridae, Cuculidae, and Tyrannidae) in a single locality. Four molecular markers, namely, the small (SSU) and large (LSU) subunits and internal transcribed spacers (ITS1 and ITS2 plus 5.8S) from nuclear ribosomal DNA, and cytochrome c oxidase subunit 1 (cox 1) from mitochondrial DNA, were sequenced from specimens of the three species. The newly obtained sequences were aligned with other sequences of Neodiplostomum spp. downloaded from GenBank. Maximum likelihood and Bayesian analyses based on each molecular marker revealed that the specimens of the three undescribed species of Neodiplostomum formed three independent lineages with high values of genetic divergence; thus, they are recognised herein as three new species: Neodiplostomumacatlotlii n. sp., N.papaloapanensis n. sp., and N.leoni n. sp. The new species showed morphometric differences in the body size, prosoma, opisthosoma, suckers, holdfast organ, testes, ovaries, and eggs. Our study also revealed that the three new species are distributed sympatrically in the Veracruzan biogeographical province in the Neotropical region of Mexico and use different ways of transmission to complete their life cycles.