Clonal amplification and weak genetic structuring in the rumen fluke Calicophoron daubneyi at a regional scale in the French Pyrenees.

Calicophoron daubneyi is an emerging rumen fluke of ruminants whose prevalence and geographic range have increased markedly across Western Europe over recent decades. Despite its growing veterinary importance, population-level genetic studies of this trematode have been hampered by the absence of suitable multilocus markers, with previous work relying on mitochondrial or ribosomal sequences that provide limited resolution for epidemiological inference. In this study, we developed and validated the first panel of polymorphic microsatellite markers for C. daubneyi and applied them to assess genetic diversity, clonality, and population structure in natural populations from the eastern French Pyrenees. Using Illumina-based genomic sequencing, we isolated 16 candidate loci, of which 10 robust and polymorphic microsatellites were retained after stringent quality filtering. We used these markers to genotype 187 adult parasites collected from cattle originating from seven localities spanning a maximum pairwise distance of approximately 50 km. Analyses explicitly accounted for the strong clonal amplification characteristic of trematode life cycles. We observed high levels of genetic diversity and a pronounced but variable clonal signal among sites. After attempting to correct for clonality and null alleles, genetic differentiation among populations was low and no significant isolation by distance was detected, consistent with substantial genetic exchange occurring at the regional spatial scale examined. These findings suggest that definitive host movement contributes importantly in shaping parasite gene flow within this interconnected livestock system. By providing the first validated microsatellite toolkit and baseline population-genetic data for C. daubneyi, this study establishes a foundation for future molecular epidemiological investigations of transmission dynamics, host-parasite interactions, and parasite dispersal in European livestock systems.