Ex situ breeding programs as a conservation management tool for the European ground squirrel in the Czech Republic

Habitat loss and fragmentation expose small, isolated populations to genetic drift, inbreeding, and loss of adaptive potential. Ex situ breeding and reinforcement are increasingly used to mitigate these risks, yet their genetic outcomes often remain insufficiently evaluated. Here, we assessed the genetic status and conservation value of ex situ breeding populations of the endangered European ground squirrel ( Spermophilus citellus ) in the Czech Republic. We genotyped 198 individuals from eight populations, including four ex situ breeding colonies and four free-living or reinforced populations, using 12 microsatellite loci. Ex situ populations retained moderate to high levels of genetic diversity and showed no evidence of elevated inbreeding; the highest allelic richness and heterozygosity were observed in Zoo Brno, whereas the lowest diversity occurred in the free-living population Radouč. Bottleneck analyses revealed contrasting demographic histories, with the strongest recent signal detected in the Rescue Centre Rozovy and evidence of an older reduction in Zoo Praha. Bayesian clustering, PCoA, DAPC, pairwise differentiation, relatedness estimates, and AMOVA consistently showed that genetic structure was shaped primarily by locality, founder origin, and reinforcement history rather than by management category alone. In particular, RC Rozovy and Písečný vrch formed a distinct genetic cluster, while Hluboká and Karlovy Vary showed close genetic affinity consistent with documented reinforcement history. These findings indicate that well-managed ex situ populations can preserve valuable genetic variation for future conservation translocations. However, reinforcement and reintroduction should be guided by population-specific genetic information and integrated with long-term habitat management to support the viability of S. citellus populations in fragmented landscapes.