the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 23 Apr 2026
Tree-microbe-soil interactions affecting soil organic carbon fractions in Mediterranean forest soils
Abstract. Soil organic carbon (SOC) represents a major terrestrial carbon pool, yet the processes that regulate its storage remain uncertain, particularly in water-limited ecosystems. The behavior of SOC is informed by partitioning into mineral-associated organic carbon (MAOC), considered more persistent, and particulate organic carbon (POC), which is more labile. We investigated how SOC fractions were affected by forest tree species, including Pinus halepensis (a canopy conifer), Quercus calliprinos (a sub-canopy broadleaf), and Pistacia lentiscus (an understory woody shrub) as compared to mixing of these species, focusing on shallow-soil mature Mediterranean stands. To elucidate further insights, the effects of soil physicochemical properties and microbial community were examined. Across soil samples, SOC concentrations were up to twofold higher under tree canopies compared to forest gaps with Quercus plots storing 10–30% more SOC than Pinus and Pistacia plots. SOC variation was primarily explained by POC, for which mixed plots showed increased concentrations as compared to monospecific plots. In contrast, MAOC displayed a saturation pattern (maximum ~45 g C kg⁻¹ soil), strongly constrained by clay and silt content, with apparent high saturation levels. Mixed forests supported seasonally stable microbial communities but did not consistently increase microbial diversity. Bacterial composition was shaped by microsite conditions, with soils under tree canopies harboring subsets of the more diverse forest-gap communities. Overall, despite the fact that mixed forest increased microbial richness, this effect did not propagate to affect the different soil C pools. Nevertheless, the effect of forest type on soil C pools was modulated by specific microsites and tree-species characteristics. For instance, transitioning to mixed forests could increase SOC by approximately 6.1 Mg C ha⁻¹ compared to monospecific pine forests, but this carbon is expected to primarily be stored in the labile POC pool, especially in soils near saturation.
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Status: final response (author comments only)
- RC1: 'Comment on egusphere-2026-2099', Anonymous Referee #1, 17 May 2026
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RC2: 'Comment on egusphere-2026-2099', Anonymous Referee #2, 06 Aug 2026
Overall comments: This manuscript represents a study on dryland soils with a comprehensive suite of analyses and an interesting study design. Authors examined how monospecific versus mixed forest stands affect soil and microbial characteristics with an emphasis on the idea of mineral saturation. Strengths of this paper included the interesting study design, and further, the writing and presentation of results are well done. I have no major concerns with the study overall, however, I do have some misgivings surrounding how afforestation and mineral saturation are presented. I think these are both fraught concepts, with afforestation presented as a possible natural climate solution when gains can be transient. Further, the limitations of mineral saturation can be observed in this study itself, where >100% saturation was reported for monospecific quercus stands. I think an interesting discussion could be had about where the idea of mineral saturation falls apart. I included more specific comments below.
Specific comments:
Lines 102-115: What about fire and transient gains? I feel that this discussion of afforestation gets tricky when we don't take into account altered fire regimes that can release a lot of forest carbon, and the fact that gains from afforestation can be transient. Regarding fire regimes, there is evidence that drylands dominate the globally burned area and that this will be exacerbated by climate change. See:
Ermitão, T., Gouveia, C.M., Bastos, A. and Russo, A.C. (2024), Recovery Following Recurrent Fires Across Mediterranean Ecosystems. Glob Change Biol, 30: e70013. https://doi.org/10.1111/gcb.70013.
Pellegrini, A.F.A., Reich, P.B., Hobbie, S.E. et al. Soil carbon storage capacity of drylands under altered fire regimes. Nat. Clim. Chang. 13, 1089–1094 (2023). https://doi.org/10.1038/s41558-023-01800-7Line 165-168: I understand that contrasting the C inventory of the pinus species versus annual plants is important for justifying the lack of focus on the transient annual plants, but is there a citation or methodology that can be included here for how this was done?
Line 194-198: Some greater clarity here regarding soil sampling is needed. In the sentence: "Next to each tree, we collected two samples at 0-1m from the trunk in opposite directions." - are the samples here soil samples?
Line 201: The use of sampling points is a bit vague here, after reading the whole section, it seems that it is a combination of a soil pit for undisturbed samples, and nearby sites for litter collection and soil samples for carbon and chemical analysis. It could be helpful to clarify this earlier in this section
Line 208-209: Was bulk density calculated after cores were sieved to 2mm? More specifically, was this just fine earth bulk density or total bulk density? Please clarify this.
Line 223-224: For determining SOC separately from SIC, there is usually some acid treatment to dissolve inorganic C. Was this performed on these samples? To be fair, the acid treatment can be fraught, see:
Apesteguia, Marcos, Alain F. Plante, and Iñigo Virto. "Methods assessment for organic and inorganic carbon quantification in calcareous soils of the Mediterranean region." Geoderma Regional 12 (2018): 39-48.Line 315: The use of the word "microsite" for what is essentially more specific forest positions might create some confusion for biogeochemists reading this - it did for me. For example, there is a rich and growing lit on the importance of anoxic microsites for C cycling - something occurring on micrometer scale. Authors could consider using a different word or clarifying this distinction earlier in the manuscript (especially in the abstract).
Line 422: I think the methodology for computing MAOC capacitance should be included in the methods. The paper that is cited by Six et al. also suggests that special consideration should be taken for calcium rich soils (they mention that this could lead to high uncertainty in MAOC - which I believe is present in this study). I think the limitations of this MAOC capacitance estimation in this particular soil type should be addressed
Section 3.5, starting at line 487: I agree with the first reviewer's comment that this section needs to be condensed
Section 4.2, starting at line 568: What about the very high saturation values reported for mono-specific quercus stands? How should the reader interpret such a high value, and wouldn't over 100% saturation suggest that there are important limitations to the idea of mineral saturation, even within this dryland context?
Section 4.3, starting at line 590: I found this section informative, but it was hard to follow the logical flow with all the previous work done at the site. Consider editing for clarity and breaking up the section
Line 597-599: There is a growing body of work on quantitative stable isotope probing (qSIP) which suggests that it's not just what microbial communities are present, but which ones are active that is impactful for SOC cycling. I understand that qSIP is highly specialized and difficult to employ, but could this possibly affect your interpretation of the microbial community results? This could also be brought up as a limitation
Figure 4 comment: Were vectors that were excluded from this figure (loading magnitudes <0.4) excluded just for readability? It creates a discrepancy between what is described in the text versus presented in the results. A full analysis could be included in supplemental materials. Red/blue distinction not color blind friendly
Figure 5 comment: there is no explanation of part e in the caption (or maybe it got cut off for me). The pinus logo also got lost and pushed towards panel d
Figure 6 comment: Red/blue distinction not color blind friendly. Also, something not explained by the caption are the box colors, some are a more saturated yellow than others, what is the reason for this?
Citation: https://doi.org/10.5194/egusphere-2026-2099-RC2
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This study collected soil samples from different forests (or forest stands), measured soil organic carbon and its fractions as well as related soil properties and analyzed the relationships between soil carbon fractions and soil organic carbon storage, together with the factors influencing soil carbon fractions. Overall, the study provides comprehensive data, employs appropriate analytical methods, and presents clear descriptions across all sections. The manuscript is generally well written and clearly organized. I have no major concerns regarding this manuscript, but only some specific minor comments for the authors to consider.
L26-49: The title of this manuscript is “Tree–microbe–soil interactions,” but the description of these interactions in the abstract is insufficient. It is suggested that the authors strengthen this aspect in the abstract.
L42-44: The abstract should focus on how environmental and microbial factors influence soil organic carbon and its fractions, rather than overemphasizing other indicators such as microbial diversity.
L146-148: This is not a complete hypothesis because the underlying mechanism is not stated. In addition, forest gaps receive relatively lower plant carbon inputs and therefore decomposition processes are expected to dominate. Since POC fractions are more easily decomposed, it can be hypothesized that the proportion of MAOC may be higher.
L157-158: What is the mean annual temperature? This is a more important indicator. Please provide it.
L217-219: It is usually not appropriate to define particles >50 μm as sand and those <50 μm as silt plus clay, because different soil classification systems use different thresholds (for example, in some systems particles >20 μm are defined as sand). It is recommended to instead define particles >50 μm as POM and those <50 μm as MAOM.
L378-528: The Results section is very detailed, but overly so. For example, the description of the structural equation modeling results, which was divided into four subsections and is unnecessary. The authors are encouraged to appropriately condense the text and focus on reporting the most important and central findings.
Figure 3b: It is not clear what the purpose of including the regression line of Díaz-Martínez et al. (2024) is in this context. Please provide an explanation. Also, why is the R² value negative? Please also provide an explanation.
Figure 3c: Is it reasonable for MAOC saturation to exceed 100%? Please verify.
Figure 4a: A linear mixed model for individual variables is acceptable, but it is not sufficiently comprehensive, because significance (*, **, ***) and R² alone cannot identify which factors are the most important in controlling soil carbon fraction storage. The authors are encouraged to further analyze the relative importance of predictors. In addition, the meanings of many indicators on the x-axis are unclear. For example, what do Temperature, Phosphorus, and Magnesium specifically refer to? Are they total nutrients or available nutrients? It would be better if these were clearly specified directly in the figure.
Figure 6: The structural equation modeling (SEM) lacks some connections among variables, such as paths from aboveground biomass and microbial communities to POC and/or MAOC. In addition, “Above ground biomass” should be changed to “Aboveground biomass”. What does “Soil properties” refer to? This cannot be determined from the figure. Moreover, does soil silt and clay content not belong to soil properties?