Abstract
We investigated the mitochondrial genetic diversity of Echinococcus multilocularis in red foxes from northern and northeastern Poland, and tested the hypothesis that the presence of Asian haplotypes in Poland stems from their introduction from Northeast Asia to Eastern Europe. We targeted the cytochrome c oxidase subunit 1 gene (cox1) of 263 tapeworms from 59 red foxes hunted in selected districts of Pomorskie, Warmińsko-Mazurskie, and Podlaskie Voivodships. We calculated several measures of genetic variation, and constructed median-joining haplotype networks and a RelTime timetree. In addition, a novel alignment-free computational method was applied: the 4D-Dynamic Representation of DNA/RNA Sequences combined with K-means clustering. PCR and sequencing were successful for 252 tapeworms. We identified six haplotypes, three of which were novel. The mismatch distribution was bimodal. The fixation index (F(ST)) between the European and Asian haplogroups was 0.97. Our findings suggest secondary contact between two previously isolated metapopulations with distinct genetic lineages. Clustering patterns appear to support the hypothesis that Asian variants of E. multilocularis spread into northeastern Poland as a consequence of multiple introductions of raccoon dogs from the Soviet Far East to Eastern Europe. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-40313-z.