Waterbirds disperse plant species via ingestion and egestion of seeds (endozoochory). However, our understanding about the regulating effects of seed traits, underlying mechanisms and possible (co)evolutionary processes is limited by our traditional reliance on data from feeding experiments with living waterbirds. Here, we overcome these limitations by developing and applying a new bioassay that realistically simulates digestive processes for Anseriformes waterbirds. We test three hypotheses: (1) seed survival and germination are most affected by mechanical digestion in the waterbird gizzard; (2) seed size, hardness, imbibition and shape regulate seed survival; and (3) plants growing in aquatic habitats benefit most from endozoochory by waterbirds. Experiments with 28,200 seeds of 48 plant species demonstrated species-specific seed survival that was entirely determined by digestion in the avian gizzard. Intestinal digestion did not affect seed survival but affected seed establishment (germinability and germination time) for 21% of the species. Large, hard seeds survived the simulations the best, in contrast to generally higher seed survival for smaller seeds during in vivo experiments. This mechanistically explains that small seeds escape digestive processes rather than being inherently more resistant (the ‘escape mechanism’), while large seeds are retained until fully digested or regurgitated (the ‘resistance and regurgitation mechanism’). Plants growing in wetter habitats had similar seed survival, but digestive processes stimulated their germinability and accelerated their germination more than for terrestrial plants. This indicates a relative advantage of endozoochory for plant species growing in wet habitats, possibly reflecting a co-evolutionary response related to dormancy breaking by gut passage. Simulating seed gut passage using a bioassay allowed establishing mechanisms and identifying relevant seed traits involved in seed dispersal by waterbirds. This information enhances our understanding of how animal species shape plant species distributions, which is extremely relevant now that current anthropogenic pressures already severely impact plant dispersal capacities.

We developed and applied a new bioassay that realistically simulates digestive processes for Anseriformes waterbirds. We tested the effects of different phases of simulated digestion on seed survival and germination in relation to seed traits (seed size, hardness, imbibition and shape). Experiments were performed with 28,200 seeds of 48 different plant species.