Microorganisms driving soil P turnover: exclusively site specific adaptions or core community?

The microbial potential for the processes of soil P turnover at sites BBR and LUE was harbored by not less than fifty-five distinct microbial orders (M1). In particular, the members of Rhizobiales, Actinomycetales, Acidobacteriales as well as Solibacterales and Burkholderiales were of importance.

Once the focus was on microbial P mobilizing enzymes exclusively, inasmuch as P transport systems were omitted, the relevance of Solibacterales and Planctomycetales was particularly enlarged. All in all, the latter six orders comprised seventy-three percent of the detected genes, which is a

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consequence of their overall domination at the two sites. On that note, the previously described capacity of rhizobial strains for the mineralization of soil organic-P was impressively underlined also in close to nature ecosystems. However, further species that are known from literature for their acid phosphatase activity including the genera of Pseudomonas, Bacillus, Serratia or Enterobacter (Rodrı ́guez and Fraga, 1999), did barely contribute to the P turnover in the two soils. Likewise the solubilization of inorganic-P, which is known to be performed by members of Pseudomonas, Bacillus, Rhizobium, Burkholderia, Achromobacter, Agrobacterium or Microccocus (Rodrı ́guez and Fraga, 1999), was predominantly associated to the members of Solibacterales in the two soils BBR and LUE.

Consequently, the implemented SGS approach (M1) partially verified the existing data, but more importantly unraveled the actual microbial key players in close to nature ecosystems.

Apart from the above-mentioned phyla, the impact of the remaining detected taxa to the processes of P turnover was of minor importance. From this point of view, the existence of a stable core of microbes that performs the turnover of soil P in distinct forest ecosystems sounds reasonable.

However, the relative contribution of specific taxa to the individual processes varied substantially with respect to the different soils. In accordance with diversity analysis (M1), at site BBR particularly the members of copiotrophic Alphaproteobacteria harbored the majority of investigated genes (40%). However, the soil nutrient content was not the sole factor that influenced the composition of the P cycling microbial community. After all, the soil P species composition and hence the significantly higher potential for the solubilization of mineral-P at site BBR induced a stronger contribution of Solibacterales in the latter soil, since members of Solibacterales were discovered as the major source for PQQGDH encoding genes. In addition, several taxa exclusively contributed to the soil P turnover at site BBR, while their impact was lacking in LUE. This includes members of Gemmatimonadetes, Poribacteria, Ktedonobacterales and Firmicutes.

In contrast, the P cycling microbial community at site LUE was shaped more homogeneously (M1).

Several different taxa were contributing to the individual processes of the soil P turnover in equal measure. While the prominent impact of copiotrophic taxa was dramatically reduced, the members of oligotrophic Acidobacteriales and Actinomycetales were of major importance. If the focus was exclusively on the alkaline phosphatase (PhoA, PhoD) harboring microbial community, a considerable decrease in diversity was detected from ten microbial orders in BBR to six orders with ongoing soil P depletion (M1), which is in accordance with the work of Tan et al. (2013). However, the authors reported an enrichment of members of the Pseudomonas group in P-depleted soils, while at site LUE particularly Burkholderial genes were dominating (M1). In this regard, Ragot et al. (2015) detected a deeply contrasting Burkholderial phoD gene abundance in distinct soil samples, whereby the singularity of the underlying microbial communities was determined. Taken together, these findings indicate an adaptation of the P cycling microbial community to the site specific conditions, regarding

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resource limitation and soil pH (Jones et al., 2009; Ward et al., 2009). Likewise for site LUE, several taxa including Cytophagales, Rhodobacterales and Desulfobacterales were associated to the P turnover exclusively in this soil. Thus, the occurrence of unique OTUs at the respective sites, which was already detected during diversity analysis (M2), was similarly reflected on functional level in terms of an exclusive contribution of specific taxa to the P turnover in a certain soil (M1). However, taking the restricted sequencing depth during metagenomics into account, the description of unique taxa might be misleading. On the one hand, deep sequencing of soil metagenomic DNA might reveal an even higher degree of site specific adaption, regarding the microbial community structure and traits. After all, SGS (M1) merely scratched the surface of the entire soil metagenome (Tringe and Rubin, 2005). Consequently, the impact of unique taxa on the soil P turnover could still be underestimated. On the other hand, the theory of Baas Becking (1934) assuming that “Everything is everywhere, but, the environment selects”, might apply on functional level as well (De Wit and Bouvier, 2006). Thus, exactly the same microbial community with identical traits could perform the turnover of soil P in distinct ecosystems. While the relative contribution of individual taxa could vary, unique taxa were rare if not lacking entirely.

In this regard, amplicon sequencing of P cycle associated genes brought new facts to light (M3). The quantity of obtained sequences exceeded the respective SGS results (M1) to a considerable degree (e.g. 117,161 vs. 38 phoD sequences) (M3, M1). Basically, the amplicon sequencing approach (M3) revealed a significantly higher diversity within the P cycling microbial community, since investigated genes from seventy-four different orders were detected compared to thirty-one orders in the SGS datasets (M1) of site BBR. This gap might to some extent be caused by the analyzed soil horizon (mineral topsoil or organic layer) since microorganisms are vertically highly stratified (Baldrian et al., 2012) and the forest floor is of peculiar importance for ecosystem P nutrition. However, the severely enhanced sequencing depth (M3) might still be the decisive factor in this respect. One the one hand, the microbial key players of the P turnover that were detected during metagenomics (M1) were also verified during amplicon sequencing, while moreover a tremendous number of genes from rare species were discovered with a low frequency (M3). In this regard, the majority of microbial orders (70%) that contributed to the P turnover at site BBR, had a low abundance (<1%) related to the total P cycle associated community. This weak, however important contribution of diverse taxa remained undetected during metagenomics (M1).

Still, it has to be taken into account that the covered microbial diversity strongly depends on the database background. As already discussed for the microbial community composition at the respective sites, also on functional level the fungal contribution to the turnover of soil P is certainly underestimated in the SGS datasets of sites BBR and LUE (M1). After all, amplicon sequencing

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detected the contribution of eight Ascomycotal and Basidiomycotal orders to the mobilization of phosphomonoesters at site BBR (M3).

In conclusion, since amplicon sequencing was merely performed for one soil type (M3), the question regarding the existence of a stable core of P cycling microorganisms in distinct ecosystems cannot be answered ultimately. However, the latter approach demonstrated that the composition of the P cycle associated microbial community is more complex than it appeared during metagenomics. Certainly, the initially detected microbial key players are of major importance in terms of ecosystem P nutrition, since they perform the bulk of soil P mobilization. In contrast, the majority of low-abundant taxa presumably is part of the rare-biosphere. While their growth strategy is preferably K-selected, these taxa are substantial contributors to ecological resilience instead (Lynch and Neufeld, 2015).

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