Large-scale Transportation Infrastructures (LTIs; roads, railways, etc.) are among the main determinants of landscape fragmentation, with strong impacts on animal dispersal movements and functioning of metapopulations. Although the detection of the impacts of LTIs is now facilitated by landscape genetic tools, studies are often conducted on a single specie, while it is acknowledged that different species might react differently to the same obstacle. Multi-specific approaches are thus required to get a better overview of the impacts of human-induced fragmentation, especially in landscapes crossed by multiple LTIs whose impacts can accumulate. We surveyed two vertebrates species (the grass snake Natrix helvetica and the midwife toad Alytes obstetricans) and two insect species (the butterfly Maniola jurtina and the ground-beetle Abax parallelepipedus) in a landscape fragmented by six LTIs: a motorway, a railway, a country road, a gas pipeline, a power line and a secondary road network. Using multiple linear regressions and commonality analyses on both classical and hierarchical genetic distances computed over reduced spatial scales, we showed that 38% of the overall explained genetic variability across all species was due to LTIs. While the butterfly was seemingly not impacted by any LTI, the genetic structure of the three ground-dwelling species was mostly influenced by roads, motorway and railway. LTIs, and especially roads, mostly acted as barriers to gene flow, barrier effects accounting for 85% of the overall variance in genetic distances explained by LTIs across species. Although the power line did not affect any studied species and the gas pipeline only impacted gene flow in the ground-beetle through forest fragmentation, other LTIs systematically affected at least two species. Importantly, we showed that some LTIs could somehow promote gene flow, embankments probably providing favourable habitats for vertebrate species. Considering the high variability in species response to LTIs, we argue that drawing general conclusions on landscape connectivity from the study of a single species may lead to counterproductive mitigation measures and that multi-species approaches are to be more systematically considered.

Patterns of gene flow across multiple anthropogenic infrastructures: insights from a multi-species approach