Summary 1.While freshwater systems provide important goods and services for society, they are threatened by human activity. Fragmentation is one of the most serious ecological concerns in the riverine environment. 2. Historical and cultural values may conflict with nature restoration. Here we use the Zwalm sub-basin (Scheldt basin, Belgium) as a case study for reconciling the restoration of the native fish fauna with the preservation of historical water mills (320-1000 years old). 3. We assessed the genetic structure of a barrier-sensitive species, the three-spined stickleback Gasterosteus aculeatus , to estimate the impact of fragmentation on a local to catchment scale. We show how population genetic approaches may be used to generate guidelines for restoration and management, and advance the science of river restoration. 4. Dispersal was lower in above-than in below-mill populations, and water mills provoked an average loss of almost 4% of the genetic variation. This loss accumulated to 40% over the entire system (~23 km, 13 barriers). The impact of individual mills strongly increased with upstream distance and water mill height. One mill provoked significant genetic differentiation, despite the presence of a fish passage. 5. This detailed picture of the genetic connectivity in stickleback is indicative for the basin's depauperate fauna. Many species share the same migratory pathways and barriers to dispersal. The physical properties of the water mills are likely to have similar effects on species with a similar genetic structure to stickleback. 6. Synthesis and applications . Population genetic studies may be particularly useful during the planning of river restoration and associated ecological studies. In the case of the Zwalm sub-basin, we propose a number of management actions, such as building new fish passages and translocating individuals to above-mill populations. These will counter the negative impact of the water mills on the genetic variation in aquatic fauna, whilst retaining their cultural-economical value and limiting the restoration costs. Simulations suggest that reassessment of stickleback genetic structure after a decade should reveal whether or not restoration actions have been effective.

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