Final Report Summary - AMFGENDIV (Environmental heterogeneity and AMF genetic diversity) Arbuscular mycorrhizal fungi (AMF) form mutualistic symbiosis with roots of the majority of terrestrial plants. They are coenocytic, known to harbour genetically different nuclei. However, the dynamic and maintenance of this genetic diversity are unknown. We have recently found two important processes that are likely to play a role in AMF: a) single spores do not necessarily inherit the same genetic material due to unequal segregation of nuclei during spore formation; b) mixing of nuclei between different individuals can occurs through genetic exchange. Under which circumstances these mechanisms could play a role in the maintenance of genetic diversity has been poorly studied and their occurrence in other AMF populations and species is unknown. Recent investigations suggest that environmental heterogeneity could be a process for the maintenance of genetic diversity and need further investigations. Additionally, studying how plants respond to the dynamic of AMF genetic structure could allow us to understand how plants and fungi co-evolve. The three objectives of the proposed project are to 1) test effect of environmental heterogeneity on AMF genetic structure; 2) determine whether segregation and genetic exchange occur in other AMF populations and species; 3) compare our results with field data. It appears clear that understandingthe dynamic and the maintenance of genetic diversity of AMF is essential for understanding the ecological importance of this very common symbiosis, for its application in agriculture and environmental management, and for understanding how organisms evolve and co-evolve over long periods of time in a mutualistic environment. This project is conduct with the group of Prof. Bever at Indiana University (US) and with the group of Prof. Sanders at Lausanne University (CH).To test the effect of environmental heterogeneity on AMF genetic structure we experimentally manipulated environmental heterogeneity in in-vitro system by using two different plant species with the following experimental design. One (to test for segregation) or two (to test for genetic exchange) AMF individuals grew in three different plant treatments at the same time: one with the first plant species, one with the second plant species and one with both plant species. We used a total of 3 plant species and 3 AMF individuals. An AMF individual correspond here to all the progeny starting from one single spore. Such an experiment has never been done before because of the difficulty toestablish large in-vitro plates without contaminations and because of the difficulty to grow on a same plate different plant species. With a common effort with another postdoc in Lausanne, we successfully obtained a large number of those plates without contaminations. However, thesuccessful establishment of those plates took a long time and we did not have time to carry out the molecular analyses. This experimental design will be used in the future in Lausanne, in collaboration with other PhDs and postdocs. We know now how to set up this experiment and we will be able toadd more variable to get an even more complete study.To determine whether segregation and genetic exchange occur in other AMF we used two new AMF species (S. fulgida and G. claroideum) that came from local field in Indiana and that were previously used in ecological studies in the Bever lab. We established single spore lines in pot cultures from those AMF species to test the effect of segregation in those AMF species on plant growth. After having established a large number of single spore lines, we successfully get a sufficient amount of single spore lines to start greenhouse experiments. We inoculated those AMF lines with two different plant species. Because those AMF were local fungi, we decided to use local plants as well, toconduct a more ecological study. We analyzed plant growth response and fund significant effects of segregation in those two new AMF species.Another objective was to establish new AMF in in-vitro cultures. We went to the University of West Virginia to meet with researcher having successfully established other AMF species in in-vitro plates. We learnt the protocol to decontaminate AMF spores coming from soil and we obtained one other AMF species in in-vitro system (G. clarum). This new AMF culture came from one single spore from soil, meaning that we were sure that the effect we looked at came from the geneticdiversity of a single spore. Because those cultures were ready to work with well before our plans, we decided to carry out experiments that were not initially planned for the outgoing phase of the grant. We established single spore lines from the initial culture to test for segregation. Moreover, we performed a change of host on those single spore lines and we measured the phenotypes of the AMF lines growing with different hosts. We then extracted DNA. Additionally, we manipulated the number of single spores that will start a new generation in order to study the effect of the initial amount of genetic diversity on the growth success of the fungi. We found clear evidence of a plant host effect on the growth of the different single spore lines. We found that the initial number of spores is critical for the growth of the fungi. We did not found clear evidence of segregation with the molecular markers we used.New studies by the group of James Bever, and by other groups, highlighted very recently interesting features dealing with resources allocation between plants and fungi. It has been shown that a single plant species is able to allocate more resources to the best mutualistic fungi when the “good” and “bad” AMF species were spatially structured. However, all those studies have been performed at the species level. The proposal we submitted aims to understand the genetic of AMF at the individual level and the consequences of this intra-individual diversity on plants and on coevolution between plants and AMF. We, therefore, decided to study preferential allocation at the individual level in three innovative greenhouse studies. The objectives of those new experiments are in line with our proposal (coevolution between AMF and plant, structuration of the environment, consequences of AMF genetic diversity at the individual scale). We carried out a large greenhouse experiment withthe three in-vitro AMF individuals coming from Switzerland. The two host plants were rice and leeks because those plants have been previously used in the group of Ian Sanders and gave interesting results. We manipulated the number of AMF individuals (one, two or three) and the spatial structure of AMF for the treatment with more than one individual (either mixed together in soil or separatedby tubes). We also performed a phosphate treatment. We found the first evidence of an additive effect of AMF genotype on plant growth. We also found a plant effect and a phosphate treatment effect, as well as significant interaction among the different factors of the experiment (plant species, phosphate treatment, AMF isolates).We decided to do a plant growth assay experiment (full cross experiment) with the material of the experiment explains above. This experiment allows us to know whether selection has occurred on AMF after the first generation of growth. Moreover, this study allowed us to investigate the effect of environmental change in time, which was one of the main objectives of the proposal. We found positive and negative feedbacks between AMF and plants. Moreover, we found evidence that selection had occurred at AMF level. Those results are important to understand the interaction between plants and fungi and can help us to understand how plants and fungi have co-evolved for more than 400 million years. Moreover, those results have important consequences in the understanding of the dynamic of AMF nuclei and on the maintenance of the genetic diversity at the AMF individual scale.Contact: Dr Caroline Angelard becomeascientist@yahoo.com; Prof. Ian R. Sanders ian.sanders@unil.ch
