In nature, most organisms interact simultaneously with multiple mutualistic species (e.g., plants with pollinators, symbionts, and ants), however mutualism studies have traditionally focused on bipartite interactions between a single partner and host. Bipartite studies will underestimate the importance of mutualism for ecological and evolutionary dynamics if synergism occurs among functionally different partners, and overestimate it if partners are in conflict. In a recent synthesis paper, we merged two disparate research approaches to define and provide a predictive ecological framework for the Multiple Mutualist Effects – MMEs – that occur when a species interacts with multiple partners. Here, we take the next step of asking how MMEs impacts the evolution of mutualisms. Using the tripartite association between arbuscular mycorrhizal fungi, nitrogen-fixing bacteria, and a model legume (Medicago truncatula), we grew >200 plant genotypes in a factorial experiment independently manipulating the bacterial and fungal symbionts. We then collected fitness and trait data to examine the impact of multispecies mutualisms on (1) fitness of the participants, (2) fitness conflict or alignment among partners, and (3) the strength, direction, and alignment of selection on mutualist traits.
We found that the MMEs had important consequences for fitness of participants, fitness alignment among interactors, and selection on host traits. For example, fitness alignment between N-fixing bacteria and the plant was achieved only in the presence of mycorrhizal fungi (rhizobia_fitness*fungi: F1,342=23.3, P<0.0001). When plants with and without mycorrhizal fungi were examined separately, we found that without the fungus fitness of rhizobia and plant were not correlated (t1,168=0.8, P=0.41) but became positively aligned with fungi (t1,174=6.3, P<0.0001). In addition, genotypic selection analysis showed that the presence of mycorrhizal fungi impacted selection on some host traits and not others. For example, selection on both rhizobia and host plants favored increased aerial shoot branching (rhizobia: F1,288=27.8, P<0.0001; plant: F1,305=148.7, P<0.0001). However, mycorrhizal fungi influenced selection strength exerted by the host plant (branch*fungi: F1,305=14.6, P=0.0002), but not by the rhizobia. In contrast, the fungus did not impact selection on flowering time, but the direction of selection did differ between the rhizobia and the plant with selection for earlier flowering times exerted by plants (F1,305=13.6,P=0.0003), but not by rhizobia (F1,288=2.1, P=0.15). Taken together our results illustrate that complex biotic environments, such as those generated by MMEs, can have important consequences for the evolutionary trajectory of mutualisms and may play a key role in avoiding mutualism breakdown through increased fitness alignment among partners.