Abstract
Reintroduction programmes are increasingly used to restore threatened freshwater fishes, yet evaluating their success remains challenging during the early stages of population establishment. Genetic monitoring is commonly used to assess diversity but rarely provides direct evidence of natural reproduction. Here, we investigated natural reproduction following reintroduction in the endangered Zingel asper, a benthic fish endemic to the Rhône River basin. Between 2006 and 2015, more than 25,000 captive-bred individuals from two source populations were released into the Drôme River (France). Using 54 microsatellite loci, we assessed genetic diversity, effective population size, relatedness and inbreeding, reconstructed parentage relationships, and evaluated admixture among source lineages through hybrid assignment analyses. Wild source populations differed markedly in genetic diversity and effective population size, whereas captive propagation reduced genetic diversity and increased relatedness and inbreeding. Despite these effects, the reintroduced population exhibited higher allelic richness than any captive cohort. Parentage analyses provided direct genetic evidence of natural reproduction by identifying individuals whose genetic origin could not be explained by the captive breeding pedigree. Hybrid assignment analyses independently confirmed admixture between source lineages. These results show how complementary genetic approaches can provide early evidence of reintroduction success and support adaptive management of threatened fish populations.