Mutualism is wide spread in nature and significantly impacts ecosystems. However, the principles governing its evolution have proved elusive. The rhizobium-legume symbiosis is one of the most sophisticated mutualistic interactions, as it results in the formation of a novel organ, the root nodule, where rhizobium is hosted intracellularly as nitrogen fixing ‘organelles’. These are named symbiosomes and produce ammonia from air.The rhizobium legume symbiosis evolved shortly after the rise of the legume family; 60 million years ago. However, by convergent evolution it also evolved more recent in the non-legume Parasponia. Ever since the discovery of Parasponia as the only non-legume that independently evolved the nodule symbiosis with rhizobium, it has intrigued the scientific community. It has been clear that this ‘bridging species’ will provide insight in how this unique symbiosis could arise during evolution. Further, it can teach us how to transfer this important agricultural trait to non-legume crops. However, it is first now that we can fully exploit the potential of this unique genus. Major insight in molecular mechanisms underlying the rhizobium legume symbiosis has been obtained by studying model legumes. This has made the rhizobium legume symbiosis one of the best understood mutualistic interactions. This insight can now be exploited to determine the evolutionary trajectory of the Parasponia rhizobium symbiosis, and to identify the genetic constraints of this interaction. Further, the revolution brought about by so-called next generation sequence technologies has made it now possible to cost efficiently sequence genomes of plant species with key positions in rhizobium nodule evolution.The overall objective of this project is to identify the evolutionary trajectory underlying rhizobium nodule evolution by using Parasponia. To validate the findings I will copy this evolutionary trajectory in Trema; the non-nodulating sister genus of Parasponia.