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Heterakis gallinarum is one of the most recurrently diagnosed nematodes within the gastrointestinal tract of galliform birds. In the present study, we investigated the genetic diversity of 96 H. gallinarum collected from free-range chickens from different localities in Tunisia. We assessed phylogeny and genetic variability using the internal transcribed spacers (ITS) of the nuclear ribosomal DNA and the mitochondrial cytochrome c oxidase subunit I gene. Haplotype and nucleotide diversities indicate that H. gallinarum is a species with extremely low genetic diversity. Based on the networks and phylogenetic trees, there was strong support for the absence of significant geographical structuring among the H. gallinarum populations in different localities in Tunisia. Mismatch distributions and neutrality tests of both genetic markers suggest that at least one expansion event occurred in the population demographic history of H. gallinarum. Our data showed a lack of population structure using the pairwise fixation index (FST) and an extensive gene flow. It indicated that the most likely major contributor to the low genetic diversity and gene flow is the movement of the parasitized birds; in other words, the frequency of the poultry trade considerably affects patterns of worm gene flow. [ABSTRACT FROM AUTHOR]

• Novel and common molecular markers support the monophyly of F. hepatica from Tunisia and Algeria. • Pepck multiplex PCR patterns could not differentiate between Fasciola species. • Sequence analysis of pepck revealed high polymorphism, length variation, within this intronic marker. • Nuclear and mitochondrial markers revealed an important gene flow between Tunisian and Algerian populations of F. hepatica. • The combination of nuclear and mitochondrial sequence analysis is still the best method to distinguish these taxa. Fasciolosis is a foodborne trematodosis characterised by a worldwide distribution. Various approaches have been developed for the study of the causative agents of this parasitic infection: Fasciola hepatica , Fasciola gigantica and the aspermic intermediated forms (hybrid and introgressed). In the present study, novel and common molecular markers (pepck and pold, ITS, CO1, ND1 and CO1-trnT-rrnL) were used to characterise Fasciola flukes from the Tunisian-Algerian border, to estimate the gene flow between these populations and to evaluate the reliability of different molecular markers. All nuclear and mitochondrial markers, apart from pepck, supported the monophyly of the studied flukes identified as F. hepatica. Multiplex PCR for pepck revealed three different genotypes corresponding to F. hepatica (pepck-Fh), F. gigantica (pepck-Fg) and the aspermic Fasciola flukes (pepck-Fh/Fg). Sequence analysis of pepck revealed high polymorphism, length variation, within this intronic marker. The observed inconsistencies were due to the position of the forward primer within the intronic region. Pepck sequences showed different level of heterozygosity and homozygosity with length polymorphisms in the introns. Pepck multiplex PCR patterns could not differentiate between Fasciola species. All studies based on only pepck multiplex PCR with mitochondrial markers should be revised. Nuclear and mitochondrial markers revealed an important gene flow between Tunisian and Algerian populations of F. hepatica. The combination of nuclear and mitochondrial sequence analysis is still the best method to distinguish these taxa. Effective measures are needed in order to better control cross-country illegal trade of vector. [ABSTRACT FROM AUTHOR]