Integrative approach for discovering of the new species within the genus Allocreadium Looss, 1900 (Trematoda: Allocreadiidae) and framing of biogeographical hypotheses for the genus.

In July 2012 new Allocreadium species was isolated from Carassius gibelio caught in the Arsenyevka River, Primorsky region, Russia. Analyses on the morphometrics and internal organs' topology revealed that these worms are morphologically closest with A. isoporum but both species are independent according to high genetic distances based on the 28S gene fragment (5.434±0.0073%). Unlike A. isoporum found earlier in Europe, the new species named A. pseudoisoporum sp. nov. has a shorter body length and the vitellarium not reaching the posterior end of the body at some distance and its anterior border is on the level of the ventral sucker. Allocreadium pseudoisoporum sp. nov. differs from seven species previously found in the Russian Far East with the following features: smaller size of the body, suckers' ratio, range values, and topology of internal structures. Newly localities in the Pavlovka River and the Artyomovka River were discovered for A. khankaiensis. Morphological variability of the worms from the Pavlovka River was observed in comparison with A. khankaiensis from the Komissarovka River. Using scanning electron microscope, we examined external surfaces of three species (A. pseudoisoporum sp. nov., A. khankaiensis, A. hemibarbi) and observed structures reminiscent sensory receptors. This study was aimed to describe species diversity of allocreadiids inhabiting the south of Primorsky region, and to clarify phylogenetic relationships between the species from the genus Allocreadium Looss, 1900 using molecular genetic methods. The phylogenetic Bayesian tree based on the 28S gene showed a clear separation of ten Allocreadium species and confirmed the validity of A. pseudoisoporum sp. nov. Allocreadium pseudoisoporum sp. nov. is most similar to A. gotoi (genetic distances - 3.578±0.0051% in 28S, and 18.777±0.0149% in cox1), and represented the earliest divergent lineage in Allocreadium clade on the phylogenetic tree based on the 28S gene, thereby indicating its proximity to the ancestral node. Also, dichotomous keys for 25 Palearctic species of Allocreadium were prepared based on the morphology of the adult worms.