1. Do urban and rural soils differ in their abiotic properties and in R. nudiflora plant traits (e.g., biomass, root shape)?
All soil characteristics were significantly different between the most extreme environments RS and DUS (Table 1). Particularly, we found that DUS showed lower N concentrations when contrasting against the other environments (64% and 49% lower than RS and OUS, respectively) (Table 1; Fig. 2a). P was the only macronutrient found enriched in DUS, showing 1.7 and 2.2 times higher concentrations than in RS and OUS, respectively (Table 1; Fig. 2b). For RS and OUS, both N and P concentrations were similar between environments (Fig. 2a, b). K concentrations were the highest in OUS (12.3 and 2.3 times higher than DUS and RS) while DUS presented the lowest K concentrations (Fig. 2c). Similarly, OUS also showed the highest concentration of Ca and Na. OUS and RS showed a similar pH, but higher than the recorded in DUS (Table 1).
When exploring differences in plant attributes among environments, we found that plants growing in OUS showed the lowest total biomass, but we did not find differences between RS and DUS (Table 1). Above-ground (shoot) biomass in RS was not different from OUS and DUS; however, plants growing in DUS showed 1.34 times higher shoot biomass than plants growing in OUS (post-hoc Tukey HSD test; Table 1). A similar pattern was found in plant height, and number of branches (Table 1). When below-ground (root) traits were evaluated, we found that in general, plants growing in urban environments (i.e. DUS and OUS) were significantly smaller in all root attributes than plants growing in RS (Table 1). Particularly, when contrasting the two more extreme environments, DUS vs RS, we found that DUS plants had 82% less root biomass, 45% less root volume, 72% shorter roots, and 23% less primary roots than RS plants (Table 1). Meanwhile, no differences were detected in root dry biomass between plants collected in OUS and DUS. Nevertheless, we detected that DUS plants have higher root volume (60%) but less and shorter primary roots than OUS plants (Table 1).
Contrasting patterns of covariation within soil and root attributes were detected among urban (DUS and OUS) and rural (RS) environments. The first principal component from the PCA including soil attributes (PC1soil) accounted for 62.9% of the variation and showed that pH, N, Ca, K, and Na covary positively among them and negatively with P concentration (Table S1; Fig. 3a). A visual inspection of ellipses in the biplot (Fig. 3a) illustrate contrasting variation between DUS, OUS, and RS environments, placing DUS soils in the higher extreme of the PC1soil where higher P concentration are present while other macronutrients are scarce (DUSPC1 soil = 2.39 ± 0.12a; OUSPC1 soil= -2.00 ± 0.15c; RSPC1 soil = -0.38 ± 0.18b; Fenvironment2, 57 = 218.71, P < 0.001).
The first principal component of the PCA including root attributes (root length, number of primary roots, root volume) was interpreted as a new variable describing root size (PC1root size; Table S2), which accounted for 64.3% of the variation. Based on this new variable, we detected that rural plants have the biggest roots and urban plants the smallest ones; in particular, plants from OUS produced the smaller roots (RSPC1 root size = 0.91 ± 0.16a; DUSPC1 root size = -0.19 ± 0.11b; OUSPC1 root size = -0.69 ± 0.08c;F2, 295 = 65.27, P < 0.001; Figure 2b). On the other hand, we also found a contrast between environments in root shape represented by PC2root shape(accounting for 25.1% of the variation; Table S2). In particular, we found that plants growing in DUS have a higher number of short and thicker primary roots than roots in OUS and RS (DUSPC2 root shape= -0.49 ± 0.68b; OUSPC2 root shape= 0.29 ± 0.06a; RSPC2 root shape= 0.23 ± 0.10a; F2, 295 = 30.38, P < 0.001). This indicates a contrasting root architecture, where DUS and OUS roots are smaller than RS roots, but DUS roots are shallower than the ones in RS and OUS (Fig. 3b).
2. Do R. nudiflora’s AMF-colonization rates, spore density, and diversity vary between rural and urban environments?
Root AMF-colonization rates in RS plats were 2.6 and 1.9 times higher than in DUS and OUS plants, respectively (Table 1; Figure 1d); however, DUS and OUS AMF-colonization did not differ significantly. Although spore density, richness, and diversity were higher in RS, it was not significantly different from DUS and OUS (Table 1; Fig. 2e, f). Furthermore, a PERMANOVA did not reveal differences in community composition between environments (R2 = 0.0029,F 2,55 = 0.08, P = 0.941). Neither spore richness, diversity, and density were associated with any soil chemical attribute (results not shown), except for a negative effect of P on spore diversity (β = -0.004, R2 = 0.093,F1,58 = 5.95, P = 0.0178).