Filtering of mutualistic partnerships in the field
Many theoretical and empirical studies have shown that stabilizing mechanisms maintain mutualistic interactions in environments with a high genetic diversity of mutualistic partners, including cheaters (Archetti & Scheuring, 2013; Kiers & Denison, 2008; Kiers et al., 2007, 2003; West et al., 2002ab). However, to our knowledge, this is the first study to reveal host filtering for genetic variants under field conditions through explicit comparison between colonized symbionts and the regional community pool. In our findings, Frankia communities in host individuals were constructed by smaller numbers of Frankia OTUs than the number of Frankia OTUs in the surrounding environments (Fig. S1, 2, S1). Moreover, the comparison with the null models showed that the Frankia communities in the host plant were not chosen from soils by chance (Fig. 2b). As a result, symbiont communities in nodules were relatively constant across host plant individuals. SeveralFrankia OTUs were detected from both rhizosphere soils and root nodules (e.g., OTU01, 02, 03, 25, 26, and 28; Fig. 1) and, while these OTUs should have infection ability, OTU 25, 26, and 28 infected only a few host individuals. In addition, even though the occurrences of OTU01, 02, and 03 were not frequent in rhizosphere, it was constantly found from nodules of most trees. Thus, the differences in bacterial assembly between roots and rhizospheres would not depend on infective or non-infective strains, but filtering mediated in determining symbiont members from the genetically diverse Frankia pool.
While filtering forces are likely to regulate symbiont assembly into a host plant in a natural system, most alders interacted with phylogenetically different OTUs (i.e., OTU01, 02, and 03; Fig. 1, S1). This may be a result of genetic variation in multiple bacterial functions. Nitrogen fixation is the fundamental function of nodulating bacteria including Frankia bacteria, promoting not only the growth of host plants but also survival and herbivory defense. Dean et al. (2014) found that nitrogen resources from nitrogen-fixing symbionts were needed for induced herbivory resistance and artificial fertilizer did not induce the resistance. This suggests that herbivore resistance does not depend on the amount of total nitrogen supply but on specific nitrogen forms, such as amides and ureides, provided by nitrogen-fixing symbionts. Nitrogen fixed by associated rhizobacteria, includingFrankia , can be stored in nodules and transported to aerial parts as these specific forms (Berry et al., 2011; Dean et al, 2009; Schubert, 1986). Furthermore, Frankia bacteria have some other plant growth promoting functions, such as solubilization of inorganic phosphate (Sayed et al., 2002) and the synthesis of plant hormones (Péret et al., 2007) and siderophores (Tisa et al., 2016). Nouioui et al. (2019) discovered genes encoding these functions from some Frankiastrains in their in silico genome analyses and detected variation in the genes among the Frankia strains. The symbioses with genetically diverse microsymbionts could be explained by the complimentary effects of genetic variation in these multiple functions of Frankia strains on host performance.
In addition, some of the Frankia strains that were obtained in a previous study, which focused on host seedlings (Kagiya and Utsumi, 2020), were not detected in the root nodule samples of most host trees in this study (Fig. S1). This may be explained by the following: (1) the above strains might be extremely rare genotypes, (2) genetic structure of Frankia might be differentiated in small spatial scales, and (3) the different communities of mutualistic partners might be constructed among host ages.