Discussion
Our aim was to study the effects of varying fungal relatedness on nutrient transfer and network formation between host roots using both whole-plants and in-vitro root culture setups. Using a whole-plant system, we found that increased intraradical colonization of the focal plant was associated with a partner plant inoculated with a non-selfing, less related fungal strain (Fig. 2a). While this difference did not significantly affect overall plant biomass (Fig. S4), it suggests that fungal competition underground may be stimulating increased fungal colonization. This is in line with past work showing an increase in intraradical fungal abundance when a plant is inoculated simultaneously with several mycorrhizal fungal species (Jin et al. 2013).
Because accurately quantifying extraradical hyphal abundance in soil-based systems is notoriously difficult (Fortin et al.2002), we further tested this idea using a three-compartmentin-vitro setup in which we could analyze the architecture of the extraradical network and harvest it in its entirety. Here, we also found that a decrease in fungal relatedness between non-selfing fungal strains was associated with an increase in extraradical fungal growth (Fig. 3a). Specifically, we found that less related, non-selfing fungal strains formed larger extraradical networks between plants compared to networks of the same strain (Fig. 3a). We did not find a statistically significant effect of relatedness on intraradical colonization in thein-vitro system (Fig. S2a&b).
We also found that changing relatedness changed the growth strategy of the fungi. Non-selfing fungal combinations were associated with higher investment in extraradical growth compared to intraradical growth (Fig. 3b). Similar results have been found in competition assays using both different species of arbuscular mycorrhizal fungi (Engelmoer et al.2014) and different species of ectomycorrhizal fungi (Hortal et al.2016). In these cases, it was suggested that allocation to growth in the soil, rather than inside the root, could help maintain a competitive edge of fungi. More generally, theory predicts that low genetic relatedness among parasites in hosts, for example, increases competition, and favors faster growth and higher virulence (Frank 1996b; West et al. 2002). We found that inoculation of roots with different species, increased the competition between the arbuscular mycorrhizal fungi, which favored an investment ratio with a bias towards extraradical (Fig. 3b), especially dense near the partner root (Fig. 4d-i).
An open question is whether a higher investment in extraradical growth changes the functionality of the network in transferring nutrients. We studied network efficiency by quantifying the transfer of quantum-dot tagged apatite from the fungal network into host roots. We added quantum-dot tagged apatite as a phosphorus source to the partner root compartment, and determined how much was transferred from the fungal network into the focal root. Unlike ‘pulse’ techniques, this new approach allowed us to quantify cumulative patterns of phosphorus transfer from the network to the host root using visual florescence in hoot roots (van ’t Padjeet al. in press.; Whiteside et al. 2019; van’t Padjeet al. 2020). We found that more quantum-dot apatite was transferred per mg extraradical fungal hyphae when the two roots were inoculated the same strain (selfing) versus transferred between the two roots in the non-selfing treatments (Fig. 5a). This increased efficiency is likely the result of anastomosis, i.e. fusion of individual hyphae of the same strain (Giovannetti & Sbrana 2001; Croll et al. 2009), in the central compartment of the selfing treatment, between A5-A5. By fusing, fungi can tap into resources of already existing mycorrhizal fungal networks, increasing the nutrient flow (Sbrana et al. 2011; Giovannetti et al.2015; Pepe et al. 2016; Novais et al. 2017). It has also been suggested that by means of fusion, arbuscular mycorrhizal fungi could create indefinitely large networks (Giovannetti et al.2004), potentially allowing for higher nutrient transport across the network per unit fungus (Fig. 5a). While only a qualitative comparison, we could visually document differences in growth strategies of the fungal network by extracting descriptive architecture data. These analyses confirmed that less related strains were characterized by dense, and more complex, fungal growth in partner root compartments, while A5-A5 networks formed denser networks in the central fungal compartment (Fig. 4d-i). This increased density in the central compartment could be the result of increased anastomosis, but more work is needed to confirm this idea.
While significantly lower compared to selfing treatments, we did find that there was also transfer of nutrients from the partner root to the focal root in the non-selfing treatments. As confirmed by qPCR in the A5-Agg treatment, this transfer is likely explained by the A5 strain from the focal root crossing the fungal compartment and colonizing the partner root compartment (Fig. S2C). By crossing two physical barriers, A5 was able to form a continuous network between the two roots, facilitating movement of phosphorus between root compartments. As past worked has confirmed that the plastic barriers used here prevent the passive diffusion of the quantum-dot tagged apatite across the plate (Whiteside et al.2019), any movement of tagged nutrients into the fungus-only and focal root compartments is via the fungal network.
This decrease in efficiency of less-related networks was translated into a growth cost for host roots (Fig. 5b). We found a significantly lower total biomass of roots when inoculated with non-selfing strains. Taken together, this suggests that competition among fungi may drive an increase in fungal size, but not in phosphorus transfer benefits to the host. This result is in agreement with past work on these fungal strains suggesting that decreasing genetic relatedness within a single host root can decrease plant growth (Roger et al. 2013). It also agrees with work showing that plant productivity does not increase with the addition of more fungal species (Van der Heijden et al. 2006; Jin et al. 2013; Boyer et al. 2015; Linet al. 2015). More fungal species can, depending on the specific plant-fungal combinations, even decrease plant size (Jansa et al. 2008; Long et al. 2010).
More generally, our data suggest that decreased genetic relatedness in fungal networks can drive changes in the overall effectiveness of the symbiosis. However, as the complexity of the environment increases, such that different strains are better able to acquire different or complementary resources, the benefits of interacting with a network of non-relatives may likewise increase (Koide 2000). Future work should aim to mimic the diverse challenges faced by plants growing in natural ecosystems as a further test of the costs and benefits of variation in symbiont relatedness.