1. Do urban and rural soils differ in their abiotic properties and in R. nudiflora plant traits (e.g., biomass, root shape)?
The literature indicates that urban soils are importantly affected by anthropogenic activity (Wiseman & Wells, 2005; Harris, 1991; Kayeet al. , 2006; Irwin et al., 2020). Nutrient enrichment, such as P and N (Kaye et al. , 2006), high temperatures (Menberget al. , 2013), compacted soil, and the presence of impervious surfaces (Day et al. , 2010) can lead to a reduction in plant size in urban environments (Yakub & Tiffin, 2017), as well as to a reduction of soil mutualistic interactions (Newbound et al., 2010; Irwinet al. , 2020). Our results show that DUS tends to have less nutrient concentrations than RS and OUS, except for P. These findings do not match with the general expectation that urban soils should be nutrient enriched (Wiseman & Wells, 2005; Kaye et al., 2006; Irwin et al., 2020). Our results highlight the high microenvironmental heterogeneity within cities that are under different anthropogenic management, such as parks and road median strips which have higher organic matter that could explain the enriched soil in OUS (Irwin et al. , 2020).
The difference between the two urban environments (DUS and OUS) can be attributed to DUS impermeable surfaces (Day et al. , 2010) that reduce P runoff and lixiviation, which is expected to be higher in OUS as well as in RS (Estrada-Medina, 2016). Meanwhile, the increase in P concentration in DUS may be caused by combustion and atmospheric deposition (Kaye et al., 2006). A previous study along a urban-rural transect in New York city, also found higher soil nutrient enrichment in rural than in urban soils (Zhu & Carreiro, 2004), concluding that the low N enrichment in cities was due to reduced nitrification from bacteria due to greater concentrations of heavy metals. In the same study, they explained the reduction in P in cities might be due to reduced organic mass inputs; however, this does not explain the higher P concentrations we found in DUS in this study. Soils in the region of Mérida city are poor in nutrients due to high soil permeability and lixiviation (Estrada-Medina, 2016); however, because Fabaceae species are dominant in the Yucatan forests, the presence of N-fixing bacteria on legumes root nodules could explain higher N concentration in rural areas (Rivero-Villar et al. , 2018). Nevertheless, these arguments cannot explain the park and road medians nutrient enrichment since Fabaceae species are missing or in a lower abundance than in rural areas (pers. obs.). High organic matter and bacteria in the rural soils (Estrada-Medina, 2016) could also explain nitrogen enrichment for both RS and OUS, although when considering the higher heavy metal concentrations in OUS we would not expect an increase in bacterial nitrification (Zhu & Carreiro, 2004).
Environmental contrasts between urban and rural areas are predicted to shape the phenotypic expression of functional traits (Cheptou & Lambrect, 2020). In plants, contrasting differences in several plant morphological attributes, have been reported (see Yakub & Tiffin, 2017), but no root morphology attributes have been explored. It is expected that urban conditions, such as higher temperature, soil compaction, and impervious surfaces, would promote reduced root growth due to reduced root elongation and fine root production (Day et al. , 2010). In this study, we confirmed this prediction by finding smaller and more compact roots in both urban environments, which can be explained by more compact and shallower soils (pers. obs.). However, given the connection between root functionality and nutrient and water intake, it would have been useful to evaluate secondary root properties such as diameter, length, density, and biomass. (Karliński et al. , 2014; Freschet et al., 2017), which have been found to be different between deep urban and rural trees (Karliński et al. , 2014). A previous study in R. nudiflora found that this long-lived herb reallocates carbon reserves into roots, as a compensatory mechanism, and that higher investment in root biomass favored reproductive compensation to herbivory (Rivera-Solís et al., 2012). Interestingly, for R. nudiflora , individuals living in the city, such compensatory mechanisms might not be relevant against folivory since the incidence of foliar damage is lower than in rural areas (pers. obs.), but it can play an even more important role to invade the city as a strategy to deal with strong and regular mowing due to street maintenance (around every month in the rainy season). This and other ecological contrasts (e.g. soil compaction) set the basis to explore for root phenotypic divergence and their implications on above-ground interactions (Bardgett et al. , 1998); however, there is currently no experimental research that has explored root phenotypic divergence driven by urbanization and their physiological and ecological implications. In this context, the use of common gardens or reciprocal transplant experiments will be key to explore such divergence and the importance of phenotypic plasticity (Nuismer & Gandon, 2008).
2. Do R. nudiflora’s AMF-colonization rates, spore density, and diversity vary between rural and urban environments?
In general, urban soil conditions have been shown to negatively affect fungal communities including ectomycorrhizal fungi (ECM) and AMF; however, there are still few studies focusing exclusively on AMF (Newbound et al., 2010). Most studies support our finding that plant populations growing in urban areas have lower rates of AMF-colonization than rural populations (see Egerton-Warburton & Allen, 2000; Wiseman & Wells, 2005; Tyburska et al. , 2013), while only one study found similar colonization rates between urban and rural environments in AMF (Karliński et al. , 2014). Interestingly, all these previous studies were conducted in tree species, and despite some of these studies recording multiple soil attributes (e.g. Tyburskaet al., 2013), only few have explored which soil chemical component drives the intensity of the interaction (Egerton-Warburton & Allen, 2000; Wiseman & Wells, 2005; Buil et al. , 2021). In this way, our study is the first to focus on an invasive perennial herb, and to explicitly disentangle the implication of urbanization on the functional role of macronutrients affecting this mutualistic interaction (see next section).
In relation to mycorrhizal communities, previous studies have assessed changes due to urbanization in ECM communities (Ochimaru & Fukuda, 2007) and in AMF communities (Egerton-Warburton & Allen, 2000; Cousinset al., 2003; Buil et al., 2021). In general, the trend is a reduction in AMF richness, diversity, and density of propagules in urban environments (Egerton-Warburton & Allen, 2000; Cousins et al., 2003; Ochimaru & Fukuda, 2007; Lin et al., 2021; Builet al., 2021). In contrast, we did not find differences in diversity or community composition between the urban and rural environments. Similarly, to the case of AMF-colonization rate, there are few studies that have evaluated the relevance of soil or other properties to explain changes in urban AMF communities (Egerton-Warburton & Allen, 2000; Buil et al., 2021). While Builet al., (2021) recorded multiple soil chemical attributes they did not statistically tested for any associations with AMF community parameters (e.g. diversity); however, they did find lower AMF richness and diversity in the more extreme urban soils, and that both were affected by vegetation coverage. On the other hand, Egerton-Warburton & Allen (2000) found, in an anthropogenic N deposition gradient, that enrichment in N reduced AMF spore richness and diversity. In our study, despite the detected contrast in soil chemistry between urban and rural soils, we did not find differences in either richness or diversity of AMF spores, and neither in community composition, but we did find a negative association between P concentrations and spore diversity. The lack of differences in spore density between urban and rural environments could be due to spore dispersion by vectors, such as wind, insects, birds, and pedestrians (Buil et al. , 2021). Contrastingly, Egerton-Warburton & Allen (2000) found that N enrichment due to urbanization reduced spore density. Further experiments should be designed to explore differences in AMF soil inoculum potential (Wiseman & Wells, 2005; Ramos-Zapata et al., 2011) between urban and rural soils to rule out the potential of low soil inoculum as a possible explanation for the decreased AMF-colonization rate shown in both urban environments (Buil et al. , 2021). The use of metagenomic approaches will be key to gain knowledge on how soil microbiome communities are affected by urbanization and to understand the role that particular taxonomic AMF groups might play in plant colonization and adaptation to urban environments (Unterseher et al. , 2011).