Our proximate and 13C NMR analysis show that litter of Ampelodesmos, the dominant perennial grass in the monitored grassland, contained the highest levels of cellulose, di-O-alkyl-C, and O-alkyl-C compounds, while the levels of lignin, N, carboxyl-C, O-substituted aromatic C, methoxyl-C, and alkyl-C were the lowest. This litter type decomposes relatively quickly on the ground (Bonanomi et al., 2019), but is also highly flammable and most of it remains in the plant tussock, a condition that promotes the occurrence of fire events in this grassland (Incerti et al., 2013). Previous studies have shown the significant and diverse effects of plant litter on soil physio-chemical properties and also on microbial communities (Schneider et al., 2012; Ping et al., 2019). Since litter input could affect soil ecological processes, including soil C and N cycling via litter decomposition, we observed that soil under Ampelodesmos had correspondingly lower levels of organic carbon, total nitrogen, and P, while soil Fe content was highest. This is probably due to the occurrence of summer fires, which allow low accumulation of litter and OC in the soil profile. Moreover, we generally found low bacterial and fungal taxonomic richness and diversity in the grassland soil. These results could be explained by the low chemical diversity of the litter combined with low structural heterogeneity, resulting in low trophic specialisation that does not allow the coexistence of different saprotrophic communities.
In addition, we found high abundance of Acidobacteria and Actinobacteria at the phylum level, whereas the abundance of Proteobacteria was low compared to the soil under shrub canopies. Proteobacteria are generally considered to be more copiotrophic, while Acidobacteria are considered oligotrophic in soil (Leff et al., 2015). Fierer et al., (2007) described Acidobacteria as oligotrophs that prefer poor soils with lower carbon availability. Therefore, their high proportion in grassland could be explained by the low C and N contents in the soil. In addition, the Verrucomicrobia phylum has been shown to utilise a variety of carbon compounds and may be closely linked to carbon cycling (Fierer et al., 2013), making it more adaptable to oligotrophic environments in soils (Noll et al., 2005). Indeed, our results are in agreement with those of Qiu et al., (2020), who found that the proportion of Verrucomicrobia was significantly lower in grasslands than in shrublands at different soil depths.
On the other hand, we found the highest abundance of Basidiomycota and the lowest of Ascomycota compared to the soil under shrub canopies. This is surprising considering the low lignin content of Ampelodesmos litter. The low abundance of Ascomycota in grasslands compared to shrub canopies suggests strong litter selection, as they are considered important decomposers in the early and intermediate stages of litter succession (Boanomi et al., 2019). Therefore, abundance of Ascomycota is likely promoted by more complex plant litter (Purahong et al., 2016). In detail, the genus Trechispora, which belongs to the phylum Basidiomycota, was found only in grassland soils. This saprophytic fungus is involved in wood decomposition processes, and its occurrence in grasslands rather than under shrub canopies is consistent with the results of Kirker et al., (2017), who found higher abundance of wood decomposition fungi for the exposed areas compared to the unexposed areas, and many of these species are well-studied wood decay fungi or at least the genera, with the exception of Trechispora spp, which is more abundant on decaying wood debris on the forest floor than on solid wood. Finally, we recored a high abundance of the genus Claviceps in the grassland soil, whereas it was almost absent under all shrub canopies. This result suggests a rather specific association between Ampelodesmos and Claviceps, which are known for the ergot disease infecting ~ 200 species of wild and cultivated grasses (Boestfleisch et al., 2015).
Are shrubs signatures specific?
We hypothesized that the diversity, richness, and composition of the microbial community would be altered by each shrub canopy, possibly due to the higher amount and/or diversity of litter trapped under the shrub canopy, which would enter the soil C and N cycles (Hooper et al., 2000). Our 13C NMR analysis shows high diversity within the litter characteristics of the shrubs studied compared to the grassland. We found that the litter of the evergreen, scherofillus Rosmarinus had a high content of lignin, N and di-O-alkyl C, while it had the lowest cellulose content. On the other hand, Pistacia had the highest content of O-substituted aromatic C while it had the lowest N content. Moreover, Olea had the highest value of methoxyl-C, while Myrtus litter had the lowest lignin content. Euphorbia, a deciduous species that sheds its leaves in summer to avoid drough period, had high N content associated with high carboxyl-C and methoxyl-C content. As a result, Euphorbia accumulated little OC in the soil compared to other shrubs such as Pistacia and Olea. Previous studies suggest that litter decomposition in soil can alter microbial biomass, composition and community structure by increasing substrate variability and diversity of chemical compounds, and that this can vary depending on litter quality (Meier & Bowman, 2008; Chapman et al., 2013). Accordingly, the evergreens Pistacia, Olea and Myrtus have demonstrated high C levels and low N levels in their soils, while the coniferous Juniperus and Rosmarinus enclose low C and N levels. Our results, at microbiota scale, showed that bacterial diversity was significantly higher under Juniperus and Euphorbia canopies than under Rosmarinus, while fungal diversity was significantly higher under Olea than under Myrtus. Collins et al., (2020) found that SE did not assign a "global signature" but was associated with increased, decreased, or no change in alpha microbial diversity when compared to soils from nearby herbaceous plant communities. Our data, instead, indicate that the microbiota signature among coexisting shrub species is highly specific.
The increase in Proteobacteria in shrub soils is consistent with the findings of Wallenstein et al., (2007), who found an increase in Proteobacteria in Arctic shrub soils, suggesting that these bacteria thrive in C- and nutrient-rich soils that develop under shrub canopies and exhibit copiotrophic properties. Previous work has shown that SE increases oxygenation and nutrient content in surface soil (Bragazza et al., 2015), suggesting that SE may influence the distribution of bacterial life strategies in the soil, i.e. enrichment of copiotrophic and depletion of oligotrophic bacteria. Moreover, all shrubs harbored a significant amount of Actinobacteria under their canopy. In particular, the Streptomyces genus was more abundant under the Myrtus canopy compared to the other canopies and the grassland. In this context, Qiao et al., (2017) studied microbial communities in nutrient-rich soils and found that Actinobacteria were more abundant than other microbes. It is now widely accepted that the establishment of bacterial communities in soils is not random but is controlled by specific compositional rules (Reinhold-Hurek et al., 2015), including plant species (Edwards et al., 2015). Interestingly, we found high abundance of free-living N-fixing bacteria, including the genera Allorhizobium, Bradyrhizobium, Mesorhizobium, and Neorhizobium, under the canopies of Euphorbia and Pistacia, while it was lower under the canopy of Rosmarinus. Surprisingly, the abundance of these free-living N-fixing bacteria was positively correlated with soil pH, phosphorus content and cations, while it was negatively correlated with soil Fe content, which could partially explain their distribution. Our results also showed that Rosmarinus had the highest Fe content, which assigns for it an intermediate-like level to grassland, while Euphorbia had a very low soil Fe content. Fe plays a fundamental role in all isozymes of nitrogenase, the ubiquitous enzyme involved in biological N fixation (Raymond, 2003). This contradicts the negative correlation between Fe and the abundance of N-fixing bacteria under Rosmarinus and grassland soils, which contain high amounts of mineral Fe. In this regard, it is possible that Fe is immobilized in the grassland matrix and thus become unavailable to microbes, as well as to plants.
The abundance of fungal phyla showed a marked variation among shrub canopies. Our findings are in line with earlier studies showing that the Ascomycota, which are early colonisers of litter and the major decomposers, are litter type specific (Štursová et al., 2020) and thus highly abundant under shrub canopies. The phylum Basidiomycota is generally better equipped for lignin degradation (Lundell et al., 2010); our results confirm that the highest amount of lignin was found in the litter of Rosmarinus, so the abundance of Basidiomycota is positively correlated with the lignin content in the litter of shrub canopies. Moreover, Mortierellomycota are known to be saprobic and ubiquitous, and several studies show that they have the ability to solubilize phosphorus and are associated with increasing yields and establishing symbioses with plants (Grządziel et al., 2019). In our study, Mortierellomycota showed significant accumulation mainly in soils under Pistacia, followed by Euphorbia, indicating the good quality of these soils due to the quality of their falling decomposed litter. In detail, our results showed that Myrtus was characterised by a group of fungi composed mainly of Penicillium (soil-inhabiting Penicillium), which are among the common producers of secondary metabolites in soil (Zhelifonova et al., 2010) and have an important function in the decomposition of organic matter (Frisvad & Samson, 2004). Park et al., (2020) found that most Penicillium species from soil are highly selective and unique to each plant. In our study, all shrubs do not form symbiosis with ectomycorrhiza, except Juniperus (Mejstrik & Cudlin, 1983), therefore, their presence under shrub canopies is in the form of free-living spores. The fact of the presence of these mycorrhizal fungi confirms the formation of islands of fertility under the shrub canopies, indicating that the soil is ready for vegetation succession. Furthermore, Euphorbia was characterised by the presence of a significant amount of weak saprophytes, including Aspergillus, Alternaria and Cladosporium, while Rosmarinus exclusively hosted the genus Didymella, which are opportunistic parasitic microorganisms that often exploit special conditions to colonise on plants and occasionally cause severe damage (Blancard, 2012). Euphorbia also exclusively harbours the genus Aureobasidium, a typical phyllospheric endophyte that is mainly found in fresh litter and rapidly disappears upon decomposition (Bonanomi et al., 2019). According to previous culture-based studies conducted on different tree species, the persistence of Aureobasidium in decomposed litter is unusual due to its limited competitive ability (Voříšková & Baldrian, 2013). Our finding, that different functional groups of litter stimulated different fungal taxa, suggests that fungi have a preference for certain litter types, probably because the ability to degrade specific organic compounds varies among taxa (Schneider et al., 2012; van der Wal et al., 2013).
Our results suggest that the composition of the fungal microbiota converges in part by the types of litter functional group enclosed by each shrub canopy (Reinhart & Callaway, 2006). This could be due to differences in chemical composition between litter types (Fanin et al., 2014; Schneider et al., 2012), with plant functional groups often playing an important role in explaining differences in chemical traits (Diaz et al., 2004). In contrast to our results for fungal communities, we did not find strong shifts in the community composition of bacteria. That the effects were particularly pronounced for fungal communities may not be surprising, considering that fungi play a key role in the degradation of more recalcitrant organic compounds (van der Wal et al., 2013). It could be that fungi are more specialised to certain litter types, while bacteria use simpler carbon compounds from litter and fungal degradation products and are therefore less responsive to different litter types. Our results are consistent with previous work showing that the addition of organic amendments to agricultural soils can influence fungal biomass and community composition in soils (Clocchiatti et al., 2020; Reardon & Wuest, 2016) and that leaf litter is associated with a unique microbiome enhancement (Asplund et al., 2018; Lin et al., 2019).
Co-occurrence network of microbial community
Microbial co-occurrence patterns have been analysed to assess the rules of community assembly and interaction networks in highly complex systems (Gotelli & Mccabe, 2002). Compared to that in shrubland, the microbial co-occurrence network in grassland was more complex, which might have been caused by the temporal and special continuity of connections between microbes, in contrast to shrubland, where connections might have been disrupted by the process of shrub encroachment. Previous studies have also suggested that positive correlations between nodes in co-occurrence networks of microbial communities in desert soil could be the result of functional interdependencies among microbial taxa under different environmental conditions (Neilson et al., 2017). In our study, the proportion of positive correlations was higher in the shrubland than the grassland, suggesting a high level of interdependence between microbial taxa in the shrubland. Topological parameters provide important information to understand microbial community structure (Newman, 2006; Ren et al., 2017). Higher values of network centralization and heterogeneity were observed in the shrubland than in grassland, indicate that there are many closely connected microbial modules (subnetworks/subcommunities in the overall network) in the shrubland.
Interactions between species are more frequent and intense in one module than in the rest of the community (Newman, 2006). Therefore, a small disturbance in the main module of a microbial network can have a large effect on the entire network of microbial communities (Liu et al., 2020). Moreover, higher mean degree and high closeness centrality and lower betweenness centrality could be used together to identify bacterial keystone taxa (Banerjee et al., 2018), which were mainly classified as Ascomycota, Actinobacteria and Proteobacteria in both networks. These keystone taxa could have a significant impact on microbial community structure and function (Banerjee et al., 2018). Removal of these highly connected taxa in a network would cause the collapse of the ecosystem structure and function (Saavedra et al., 2011).