3. How are AMF-colonization rates on R. nudiflora associated with soil properties, and to what extent are these associations affected by urbanization?
In our study, the recording of several soil chemical attributes and the combined use of different statistical approximations allowed us to detect association patterns between soil chemicals and the plant-AMF interaction. First, a PCA detected a positive pattern of association between a set of soil chemical properties (pH, N, K, Na, Ca) that were negatively associated with P concentrations (PC1soil). In DUS, where P concentrations were the highest, we detected a reduction in AMF-colonization as P concentration increased and N, K, Ca, Na, and pH were reduced (PC1soil). This result suggests that P plays a key role in reducing plant-AMF interaction as previously reported (Smith & Smith, 2011; Chen et al., 2020), or that the reduction on the other soil properties were mediating the interaction. Also, this result indicates that it would be crucial to distinguish the indirect and direct effects of each variable influencing the R. nudiflora -AMF interaction, using a SEM approach and considering the patterns of covariation of the chemical properties.
With a SEM approach we were able to detect and confirm that P concentration played a key role in reducing the AMF-colonization rates in R. nudiflora , as it was expected (Salvioli di Fossalunga & Novero, 2019; Chen et al., 2020; but see Treseder & Allen, 2002). Our results indicate that a negative effect of P on AMF-colonization rates was observed in DUS and RS, but while in DUS the higher P concentrations result in a reduction of the AMF-colonization rates, the lower concentrations of P promotes higher AMF-colonization rates in RS. While this explains the results found in DUS and RS, for OUS we found low P concentrations and low AMF-colonization rates, reflected on the positive effect of P concentrations on AMF-colonization in the SEM. Chen et al. (2020) found a reduction in the magnitude of the effect of P on AMF-colonization rates when native plants were competing with invasive plant species, which have a greater negative effect. It is possible that, in OUS, R. nudiflora encountered greater competition, which changed the effect that P had on the plant-AMF interaction. Furthermore, contrary to previous evidence (Egerton-Warburton & Allen, 2000; Karliński et al., 2014), we found a consistent positive effect of N concentrations on AMF-colonization rates, which can be expected in N-limited soils. Moreover, similar positive effects of P on AMF can also occur in P-limited soils (Treseder & Allen, 2002). Such positive effect of P and N on AMF-colonization can suggest that AMF are not only constrained by carbon supplied by plants but also by soil nutrient availability (e.g. N and P), and that below a certain threshold the addition of P and N can have positive effects on AMF (Treseder & Allen, 2002).
While the above studies can explain the positive effect in N among the sampled environments (i.e. DUS, OUS, RS), there is still limited knowledge to help us to understand the observed associations between K and AMF-colonization in our study (but see Benito & González-Guerrero, 2014; Garcia & Zimmermann, 2014). Even though K is a very abundant element of soil composition, it has a low availability due to strong mineral adsorption. In this way, plant-AMF association can improve K uptake, favoring salt and drought stress tolerance in host plants (Benito & González-Guerrero, 2014). To our knowledge, the functional shift in K detected in our study has not been previously observed; however, such change can be associated with K concentration (see Schreiner & Linderman 2005). Accordingly, in DUS, where K was found more limited, we detected the strongest positive effect favoringR. nudiflora -AMF association, potentially increasing stress tolerance in R. nudiflora individuals from the most extreme urban environment. Considering K in studies focusing in plant-AMF can be fundamental to understand this interaction due its direct and indirect implications mediating plant nutrition (Dibb & Thompson, 1985). Finally, the negative effects of pH detected on P, K, and N are consistent with previous reports (Wiseman & Wells, 2005; Osman, 2013), supporting the idea that the indirect effect of pH can drive, through changes in macro-nutrient availability, the plant-AMF mutualistic interaction.
Despite the fact that several studies have evaluated the effect of urbanization on the plant-AMF association (Egerton-Warburton & Allen, 2000; Wiseman & Wells, 2005; Karliński et al. , 2014; Wiseman & Wells, 2005; Buil et al. , 2021), few of them have assessed which soil chemical properties predict the intensity of this mutualistic association (Egerton-Warburton & Allen, 2000; Wiseman & Wells,2005; Buil et al. , 2021). Egerton-Warburton & Allen (2000) found that vehicular deposition of nitrogen oxides created a eutrophication gradient in which N enrichment reduced AMF root infection in a plant community in Riverside-Perris Plain, Southern California. In the same way, in disturbed landscape soils, Wiseman & Wells (2005) found that AMF-colonization in red maple roots was higher on more acidic soils in Piedmont, South Carolina. They suggest that such increment in AMF infectivity might be due to an indirect effect of acidification, reducing P availability and increasing the benefit of the association with AMF to increase P uptake; however, they did not find statistical differences in P concentrations between developed and undeveloped sites, nor a direct association between P and AMF-colonization. Finally, Builet al. (2021) found that AMF infectivity was negatively associated with subsoil compaction in a gradient of four urban sites with different disturbance levels in Córdoba, Argentina. We believe that recording several soil chemical properties is the key to disentangle complex patterns of association that would help to understand the ecology and evolution of plant-AMF mutualistic interactions. In particular, our preliminary analyses using univariate analyses only detected positive effect of K on AMF-colonization, so the use of SEM was fundamental to disentangle the hidden effects by other macronutrient.