the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 29 Jul 2026
Soil horizon development and altitudinal-vegetational zonation decouple microbial biomass from bulk soil nutrient availability across a Himalayan treeline ecotone
Abstract. Alpine treeline ecotones are environmentally sensitive transition zones where vegetation structure, soil development, and nutrient cycling interact over short spatial scales. Despite increasing attention to Himalayan treeline dynamics, the distribution of soil microbial biomass and its stoichiometric relationships across vegetation and soil horizon gradient remains poorly understood. We quantified microbial biomass carbon (MBC), nitrogen (MBN), and phosphorus (MBP), together with soil organic carbon (SOC) and total nitrogen (TN), across four altitudinal–vegetational zones and four genetic soil horizons in the Rolwaling Himalayan treeline ecotone, Nepal. Linear mixed-effects models were used to evaluate the effects of soil horizon development, vegetation zonation, and slope aspect on microbial biomass concentrations and stoichiometric relationships. Soil horizon development was the dominant control on microbial biomass distribution. MBC, MBN, and MBP declined significantly with depth (p < 0.001), with the surface O-horizon accounting for 55.7 %, 62.0 %, and 62.8 % of the cumulative horizon-wise concentrations of MBC, MBN, and MBP, respectively. Altitudinal-vegetational zonation generated pronounced non-linear patterns. SOC and TN concentrations were highest in the lower krummholz zone dominated by Rhododendron campanulatum, whereas MBC and MBN concentrations peaked in lower dwarf shrub heath zone, demonstrating a clear decoupling between bulk soil nutrient concentrations and microbial biomass. Microbial phosphorus concentrations exceeded microbial nitrogen concentrations by 3.9-fold across the study area, yielding an overall microbial biomass stoichiometry of 3.4:0.3:1 (MBC:MBN:MBP), substantially different from globally synthesised microbial biomass ratios, indicating plasticity in microbial biomass stoichiometry in stress environments in treeline ecotones. Our results indicate that microbial biomass distribution across the Rolwaling treeline ecotone is shaped by both soil horizon differentiation and altitudinal-vegetational zonation. The contrasting spatial patterns of microbial biomass and bulk soil nutrient concentrations suggest that measurements of total soil nutrients alone may not fully capture biologically active nutrient dynamics in high-elevation ecosystems.
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RC1: 'Comment on egusphere-2026-3815', Anonymous Referee #1, 21 Sep 2026
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AC1: 'Reply on RC1', Rabindra Adhikari, 05 Nov 2026
Response prepared for referee #1 comments on egusphere-2026-3815:
“Soil horizon development and altitudinal-vegetational zonation decouple microbial biomass from bulk soil nutrient availability across a Himalayan treeline ecotone."
(Note: for the clear distinction, the reviewer's comments are in light blue colour, and the corresponding responses are in automatic text color)
In this manuscript, Adhikari et al. investigated the spatial distribution and stoichiometric relationships of soil microbial biomass (MBC, MBN, MBP) across an altitudinal–vegetational gradient and four soil horizons in the Rolwaling Himalayan treeline ecotone, Nepal. This manuscript addresses a critical knowledge gap regarding soil microbial dynamics and nutrient stoichiometry in high-elevation ecosystems. The dataset combined both soil horizon differentiation and altitudinal-vegetational zonation in an understudied region (the Himalayas). A particularly interesting and well-presented finding is the empirical demonstration of a clear decoupling between bulk soil nutrient availability (SOC, TN) and microbial biomass (MBC, MBN), as well as the unusually low C:N:P ratio (3.4:0.3:1) indicating microbial stoichiometric plasticity under environmental stress. Overall, the manuscript is well-written and logically structured. However, there are several conceptual, methodological, and interpretive limitations that need to be addressed before publication. Below are my detailed major and minor comments:
R: We sincerely thank the reviewer for the careful and constructive evaluation of our manuscript and for recognizing the relevance of the study. We greatly appreciate the reviewer’s detailed comments, which have helped us identify areas requiring clarification, methodological detail, and more cautious interpretation. Specifically, reviewer comments were very helpful to minimize reflective speculations in the discussion. We have carefully considered each point and will revise the manuscript accordingly, with particular attention to strengthening the conceptual framing, clarifying methodological procedures, and ensuring that our interpretations remain appropriately supported by the data. Our point-by-point responses to all comments showing how we will revise the new version of the manuscript are provided below:
line 48, please specify the altitude information (elevation range in meters above sea level, e.g., m a.s.l.) for the treeline in the Rolwaling Himal to provide necessary spatial context for the reader.
R: We agree with the reviewer’s suggestion, and we will add the treeline elevation range (approximately 3900–4000 m a.s.l.) in the Study Area section of the manuscript to provide spatial context for the study system.
Line 66: The statement that "In high-altitude treeline regions, low soil temperature and recalcitrant organic inputs slow down the decomposition of organic matter" is somewhat too absolute and generalized. In Section 4.2, you correctly highlight that substrate quality plays an important role in regulating microbial processes. To ensure logical consistency throughout the manuscript, please rephrase this statement and briefly mention the modulating influence of substrate quality and micro-environmental variability.
R: We agree with the reviewer’s suggestion and will revise this statement. The revised text will emphasize that decomposition and nutrient mineralization in high-altitude treeline ecosystems are influenced by the combined effects of temperature, substrate quality, vegetation composition, soil properties, and micro-environmental variability with added supporting references.
Lines 101–105: The main objective is not clearly described here. Please consider rephrasing this section.
R: Thank you so much for your comment pointing this out. We will revise the final paragraph of the Introduction in the revised manuscript to clearly state the overall objective of the study and the specific aspects investigated before presenting the hypotheses (e.g. “this study aims to investigate how soil horizon development and altitudinal–vegetational zonation influence microbial biomass C, N, and P distributions and microbial stoichiometric relationships across the Rolwaling Himalayan treeline ecotone").
Figure 1: Please add the directional indicators (E for East longitude and N for North latitude) to the geographic coordinate labels on the map to ensure standard cartographic formatting.
R: We agree with the reviewer’s suggestion and will revise Figure 1 by adding directional indicators (N and E) to the geographic coordinate labels to improve cartographic clarity.
Figure 2: Please consider adding explicit elevation tick marks and values to the vertical axis (elevation axis) to make the altitudinal gradient clearer and easier for readers to interpret visually.
R: We agree with the reviewer’s suggestion. We will revise Figure 2 by adding explicit elevation marks and values (m a.s.l.) to the vertical axis to improve visualization and interpretation of the altitudinal gradient.
Figure 2 & Table 1: In the caption of Figure 2, you refer to "NE and NW aspects", whereas in Table 1, you use "NE slope, NW slope". Slope (gradient/inclination) and slope aspect (cardinal direction/exposure) are distinct topographic terms. To maintain consistency, please adopt a uniform terminology, e.g., "aspect" or "slope aspect", throughout the entire manuscript, including figures, tables, and main text.
- Thank you so much for pointing out this. We agree with the reviewer’s comment. We will revise the terminology throughout the manuscript and now use “slope aspect” when referring to slope exposure direction (e.g., NE and NW slope aspects) to ensure consistency between figures, tables, and text.
Table 1: under the "Zone characteristics" column for the UD zone, the descriptive text is set in italics. Only the Latin botanical names should be italicized. Please ensure that all Latin vegetation/species names throughout the manuscript are consistently formatted in italics, and that non-taxonomic text remains in standard font (e.g., check Table 1, line 155, line 432, line 480, among others).
R: We agree with the reviewer’s comment and will carefully check the manuscript for botanical nomenclature formatting. We will correct the italicization so that only Latin taxonomic names are italicized, while descriptive ecological terms and non-taxonomic text remain in regular font.
Section 2.3 Field Sampling: Please provide the explicit sampling depth information (depth ranges in centimeters, e.g., 0–10 cm, 10–20 cm) for each genetic soil horizon/site sampled in Section 2.3. Additionally, please consider summarizing these depth ranges clearly in Table 1 or Table 2 to give readers a complete vertical reference for the soil horizon layers.
R: Thank you so much for pointing this out. We will add the depth information of the sampled soil horizons in the Field Sampling section and will summarize the corresponding depth ranges of each horizon in Table 1 to provide a clearer vertical reference for the soil profile.
Section 2.4 Laboratory Analysis. (i)Line 170: Please briefly explain the rationale for choosing 20 °C as the incubation temperature (e.g., standard laboratory incubation protocol vs. field temperature relevance) and provide the corresponding reference. (ii)Lines 172–173: The authors state that "a total of 7.5 g of moist soil" was used. Please explain why a 7.5 g sample mass was selected instead of a more standard weight (e.g., 5 g or 10 g). (iii)Lines 188–189: You mention that "carbonates were absent from the soil." Please state how this was confirmed. Did you perform a field/laboratory test (such as dilute HCl effervescence or total inorganic carbon analysis)? If so, please specify the analytical method used here.
R: We appreciate the reviewer’s request for clarification. We will revise the Methods section to clarify that incubation at 20°C was applied as a standard laboratory procedure for microbial biomass estimation under controlled conditions, rather than to reproduce the actual soil temperature in the field. We will also clarify that the 15 g samples were weighed. The volume of organic layer soil was very large to adjust our lab equipment and revised as per the local adjustment for the lab facility (7.5 g for fumigated and 7.5 g for non-fumigated). Moisture correction was applied later in the calculation. In addition, we will specify that the absence of carbonates was confirmed using the dilute HCl effervescence test in the field.
Section 2.5 Statistical Analysis. Line 206: (i) Please assign a formal equation number (e.g., Eq. 1) to the model formula for easy reference. (ii) If I understand correctly, in the formula log10(Y) ~ (zone + aspect + soil horizon)² + (1|plot id), the notation “2” uses R formula syntax to represent all main effects and two-way (second-order) interactions, right? To avoid confusion with quadratic or polynomial transformations (e.g., squaring a variable), please explicitly clarify in the text that “2” denotes the inclusion of all two-way interaction terms among the three fixed factors.
R: We agree with the reviewer’s suggestion. We will assign an equation number to the model formula and will clarify that the “^2” notation in the R formula represents the inclusion of all main effects and two-way interactions among the fixed factors (zone, aspect, and soil horizon), rather than a quadratic transformation.
Figure 3: The abbreviations LC, UC, LD, and UD are referred to as "four topographic zones" in the caption of Figure 3, but as "four altitudinal–vegetational zones" in other parts of the manuscript. Please confirm whether both terms refer to the exact same zones. If so, please adopt a single, uniform naming convention throughout the manuscript (e.g., "altitudinal–vegetational zones"). Consider briefly describing their key topographic features (e.g., convex, depression) in the main text or summarize them in Table 1 to clarify the distinction between topographic and vegetational drivers.
R: We agree with the suggestion. The terms referred to the same four zones; however, “topographic zones” was not the most appropriate terminology because the zones were defined based on elevation and vegetation characteristics. We will revise the manuscript to consistently use “altitudinal–vegetational zones” throughout Figure captions, tables, and text.
Use of Abbreviations.The abbreviation for Total Nitrogen (TN) is defined multiple times in the main text (e.g., Line 169, Line 238). An abbreviation should be defined at its first mention and used consistently thereafter. In addition, for terms like Soil Organic Carbon (SOC), the full name is repeated in later sections even after the abbreviation was already established. This undermines the purpose of using abbreviations. Please carefully proofread the entire manuscript to ensure that all technical abbreviations (SOC, TN, MBC, MBN, MBP) are defined once upon first occurrence and used consistently throughout all subsequent text (caption of tables and figures, abstract are not included).
R: Thank you so much for pointing this out. We will carefully check the manuscript and will ensure that abbreviations such as SOC, TN, MBC, MBN, and MBP are defined at their first occurrence and used consistently thereafter. Repeated definitions and unnecessary use of full terms will be removed.
Figure 4. (i)Letter sequence: Please ensure that the lowercase letters indicating significant differences follow standard scientific convention, starting with 'a' for the horizon with the greatest mean/median value and descending progressively (a > b > c). (ii)Label placement: Please reposition all letters slightly above the upper bound of the error bars to prevent overlap and improve visual clarity. (iii)Missing annotations: In the MBC plot, the letters for the Bh horizon are missing. Please check whether this is a plotting oversight or due to data constraint, provide the missing statistical annotations, and clarify the cause if data for this horizon were excluded from the analysis.
R: We thank the reviewer for pointing this out. We will check the statistical annotations and will revise the placement of the lowercase letters to improve their visibility. Just to make it clear, the absence of the Bh-horizon annotation in the MBC panel is not a plotting omission: the Bh horizon was not present in the UD zone where soil with shallow depths occur. We will clarify this in the figure caption of the revised manuscript.
Table 2. Please correct the unit for SOC. The current notation "mgg-1 soil" lacks proper spacing. Please update it to standard format: mg C g-1 soil". Please add a space before all "±" symbols in Table 2 (e.g., replace 12.4± 0.5 with 12.4 ± 0.5) to ensure symmetric formatting throughout the table.
R: Thank you so much for pointing this out, we will correct and make the systematic formatting throughout the revised manuscript for unit and symbols. Thank you so much for pointing out in detail, it is very helpful.
Figure 5. (i)The unit for bulk soil organic carbon is given as "g C kg-1 soil" in the horizontal axis and caption of Figure 5, whereas "mg C g-1 soil" is used in Table 2 and Figure 3. Using different unit expressions across figures and tables creates unnecessary confusion. Please adopt a single, uniform unit notation across all figures, tables, and text. (ii)Statistical symbols such as R2 (coefficient of determination) and p (probability value) in the caption of Figure 5 should be italicized. Please check and ensure that statistical abbreviations and variables are consistently italicized throughout the entire manuscript.
R: We thank the reviewer for noting these formatting inconsistencies. We will standardize the SOC unit in Figure 5 and its caption to mg C g⁻¹ soil, consistent with Table 2 and Figure 3. We have also checked the manuscript and will revise the formatting of statistical symbols, including R² and p, to ensure consistent italicization throughout the revised manuscript.
Please consider increasing font size in figure 6 and figure 4.
R: Thank you so much for the comment, Figures 6 and 4 will be revised with increased font size in revised manuscript.
In Section 3.2.4, you present results from a Principal Component Analysis (PCA). However, there is no corresponding description of this statistical method in the Methodology section. Please add a dedicated subsection or paragraph detailing the PCA methodology.
R: Thank you for pointing this out. We will add a dedicated description of the PCA procedure to the Methods section. The revised manuscript text will specify the variables included in the PCA, treatment of missing observations, standardization of variables prior to analysis (we have done this), the R package and function used for the PCA, and the procedure used to visualize the resulting ordination by vegetation zone.
Line 425-426, The authors attribute the decline in microbial biomass with soil depth primarily to "increasing energetic and chemical constraints in mineral soil horizons." However, as noted in Line 423 regarding organic matter occluded within soil aggregates, physical protection mechanisms significantly limit microbial accessibility to substrate. Besides, oxygen availability decreases substantially with increasing soil depth, imposing severe aerobic metabolic constraints on subsoil microbial communities. Please broaden this paragraph to explicitly incorporate physical protection and depth-dependent oxygen limitation alongside energetic and chemical constraints when explaining subsoil microbial biomass dynamics.
R: Thank you so much for these very crucial comments on the discussion. We will broaden the discussion of the decline in microbial biomass with soil depth to include not only reduced substrate and energetic availability but also potential effects of physical protection of organic matter and reduced oxygen availability with increasing depth (e.g. including the reference from (Wilpiszeski et al., 2019; Tecon & Or, 2017). These mechanisms will be presented as potential explanations rather than directly measured processes.
Line 438, check the format of the unit “1300mm yr-1”.
R: Many thanks for pointing this out, checked and corrected as 1300 mm yr-1.
Lines 465-466, you stated that “soil nutrient concentrations (SOC and TN)”. SOC represents an organic substrate and energy source, rather than a nutrient. While TN, TP, and inorganic nitrogen forms are nutrients, carbon provides the structural/energetic framework for microbial heterotrophy. Please rephrase this phrasing and check the entire manuscript to ensure SOC is not incorrectly classified as a soil nutrient.
R: We thank reviewer for this correction. We will revise the wording to distinguish SOC from nutrients and now describe SOC primarily as an organic carbon substrate and potential energy source for microorganisms. We have also checked the manuscript for similar terminology and will revise the relevant statements for consistency throughout the revised manuscript.
Lines 475-485. (i)The authors interpret elevated qMIC as evidence for “more efficient microbial utilization” and “faster nutrient cycling.” However, qMIC is a stock ratio (MBC/SOC) and does not directly measure carbon use efficiency (a flux ratio) or nutrient turnover rate. High qMIC indicates greater immobilization of carbon in microbial biomass, which may reflect lower turnover, shifts in community composition, or differences in substrate quality, rather than faster nutrient cycling. (ii) The inference from “reduced surface litter retention and increased exposure to wind and radiation” to “greater incorporation of organic substrates into mineral soil” lacks an explicit mechanism. Exposure to wind and radiation more commonly implies loss (erosion, photomineralization, leaching), not downward incorporation. If the authors wish to argue for enhanced incorporation into mineral soil, they should articulate the intermediate pathway, e.g., photodegradation and physical fragmentation of surface litter into finer, more soluble fragments that are subsequently leached into mineral horizons and ideally support it with relevant measurements (DOC flux, litter fragment size, or isotopic source tracing). Without this, the causal link remains speculative.
R: We appreciate this correction regarding the interpretation. We will revise the interpretation of qMIC to reflect that it is a stock-based MBC:SOC ratio rather than a direct measure of carbon-use efficiency, microbial efficiency, or nutrient turnover in our revised manuscript. We will also revise the discussion of litter exposure and carbon incorporation to avoid implying a causal mechanism that was not directly measured in this study. We will describe photodegradation, physical fragmentation, and subsequent production and leaching of soluble organic compounds into mineral horizons only as potential pathways, supported by previous studies (e.g. (Brandt et al., 2009) rather than as processes demonstrated by our data.
Section4.3. The authors state “microbial stoichiometric ratios differed substantially from globally reported averages, supporting our third hypothesis that microbial communities exhibit localized stoichiometric responses to environmental conditions”. Deviation from a global average only demonstrates that the system differs from the global mean; it does not demonstrate a response to local environmental conditions. The third hypothesis concerns a response, which requires evidence that stoichiometric ratios covary systematically with local environmental drivers. A single deviation from a global constant cannot provide such evidence, because the deviation may arise from any number of causes, e.g., methodological differences, distinct community composition, site history, rather than from local environmental control. Notably, the authors later report "substantial variation in soil stoichiometric ratios both along the soil profile and across altitudinal and vegetation zones," which is precisely the kind of gradient-based evidence that could support the third hypothesis. However, this evidence is not used to support the claim here; instead, the global comparison is presented as the supporting evidence. This is a mismatch between evidence and argument. Consider reframing the comparison with the global average as a descriptive benchmark rather than hypothesis support.
R: Thank you for this important input. We agree that deviation from a global average provides a descriptive comparison but does not, by itself, demonstrate a localized response to environmental conditions. Moreover, we have this result from only one study area, we have therefore revised section 4.3 so that the comparison with globally reported microbial stoichiometric ratios is presented as a descriptive benchmark. We will discuss the observed variation in microbial stoichiometric ratios along the soil profile and across altitudinal-vegetational zones as relevant evidence while avoiding causal interpretations.
Line 515–516. The sentence starting with "Hence, in alpine ecosystems where..." is a sentence fragment lacking a main predicate. Please revise or merge it with the subsequent sentence.
R: We sincerely thank the reviewer for pointing out this sentence structural error. We will revise this paragraph as: „Similar departures from global stoichiometric expectations have been reported from cold alpine ecosystems of the Tibetan Plateau and Gongga Mountain, where microbial communities display elevated stoichiometric plasticity and strong spatial variability in nutrient allocation (Fan et al., 2021; Sun et al., 2013). Hence, in alpine ecosystems, where low temperatures, nutrient limitations, and strong seasonal fluctuations impose physiological constraints on microbial growth, microbial biomass stoichiometry may reflect local environmental conditions rather than a fixed global elemental composition. “
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AC1: 'Reply on RC1', Rabindra Adhikari, 05 Nov 2026
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RC2: 'Comment on egusphere-2026-3815', Anonymous Referee #2, 26 Sep 2026
This manuscript investigates microbial biomass C, N, and P and microbial stoichiometry across soil horizons and altitudinal–vegetational zones in a Himalayan treeline ecotone. The study addresses an interesting topic and provides a useful dataset across contrasting vegetation zones and genetic soil horizons. However, several methodological and interpretative issues need further clarification. The main concerns relate to the preparation of soil samples before microbial biomass analysis, the calculation and interpretation of relative horizon contributions from mass-based concentrations, and several mechanistic interpretations that extend beyond the variables directly measured in this study. The statistical interpretation and consistency between the results and conclusions should also be carefully checked. My specific comments are provided below.
Abstract
- Horizon contributions
The statement that the O-horizon accounted for 55.7%, 62.0%, and 62.8% of MBC, MBN, and MBP should be reconsidered. These values are based on mass-based concentrations rather than stocks. Concentrations from different horizons cannot be directly summed to represent their contribution to the whole soil profile without considering horizon thickness and soil mass or bulk density. Please revise this interpretation throughout the manuscript.
- Stoichiometric plasticity
The statement that the microbial biomass stoichiometry of 3.4:0.3:1 indicates stoichiometric plasticity is too strong. The observed ratios show differences from globally reported values, but they do not directly demonstrate microbial physiological plasticity. Please revise this interpretation.
Introduction
- Environmental stress
Please check the discussion of environmental stress. High pH is mentioned as an environmental stress, whereas the soils at the study site are described as strongly acidic. Please revise this paragraph to better reflect the conditions relevant to the study area.
- Microbial CUE
Please use caution when discussing microbial CUE because CUE was not measured in this study. The relationship between the observed biomass stoichiometry and CUE should not be presented as a mechanism directly evaluated here.
- Hypothesis 3
Please revise H3. Differences in microbial biomass stoichiometry can be tested directly, but “nutrient constraints” and “microbial adaptation” are mechanistic interpretations that were not directly measured in this study.
Materials and Methods
Soil sampling and laboratory analysis
- Soil drying before microbial biomass analysis — major concern
Please justify the use of soil dried at 40 °C for microbial biomass analysis. Drying followed by rewetting and three weeks of incubation may alter the microbial biomass compared with the original field condition. Please discuss how this sample preparation may affect the interpretation of MBC, MBN, and MBP.
Expression of soil properties
- Relative contribution of soil horizons — major concern
Please reconsider the calculation and interpretation of the relative contribution of each horizon. Concentrations expressed per unit soil mass cannot be directly summed across horizons to represent their contribution to the whole soil profile without considering horizon thickness and soil mass or bulk density.
Statistical analysis
- MBP model simplification
Please provide more details on the simplification of the MBP model. Specify which interaction terms were removed and how the final model structure was selected after the singularity problem was identified.
- Zero or negative CFE flushes
Please provide further justification for treating all zero or negative CFE flushes as experimental artifacts and assigning them as missing values. Please clarify whether these observations were checked for analytical or procedural problems before exclusion.
Results
- Figures 3–6: Please improve the overall quality and readability of these figures. Some labels, symbols, and text are difficult to read at the current size. Please increase the figure resolution and ensure that all graphical elements remain clearly legible at publication size.
Soil organic carbon and total nitrogen
- Profile contributions
Please revise this interpretation according to the comment in the Methods regarding the calculation of horizon contributions.
Microbial biomass
- Non-significant zone effect
The effect of altitudinal–vegetational zone was not significant (p = 0.259). Please avoid describing these differences as a distinct pattern and present them as descriptive trends only.
- p = 0.058
The interaction was not statistically significant at the stated α = 0.05 (p = 0.058). Please avoid referring to it as “marginally significant.”
Microbial stoichiometry
- Overall 3.4:0.3:1 ratio
Please clarify how the overall microbial biomass stoichiometry of 3.4:0.3:1 was calculated, particularly because MBC, MBN, and MBP contained different numbers of missing observations.
- Very high variability
The MBC:MBN ratio in the E-horizon shows extremely high variability (CV = 396.9%). Please discuss this variability when interpreting the approximately 17.8-fold difference from the O-horizon.
Discussion
4.1 Vertical pedogenic control
- “Biological activity”
Please revise “biological activity” because microbial biomass was measured, but microbial activity was not directly assessed.
4.2 Decoupling between microbial biomass and bulk soil nutrients
- qMIC and microbial efficiency
This interpretation is too strong. Higher qMIC indicates a greater proportion of SOC present as microbial biomass, but it does not directly demonstrate more efficient substrate utilization or faster nutrient cycling.
- Biologically available nutrient fractions
This statement is not directly supported by the measured variables. Biologically available nutrient fractions and microbial activity were not measured in this study. Please revise this interpretation.
4.3 Microbial stoichiometry and nutrient constraints
- Stoichiometric plasticity
Please revise this interpretation according to the earlier comment regarding microbial stoichiometric plasticity.
- Severe N limitation
Please use caution when interpreting high MBC:MBN ratios as evidence of severe N limitation. Nitrogen limitation and the proposed shifts toward maintenance and nutrient scavenging were not directly measured in this study.
4.4 Ecological implications
- Nutrient availability and microbial utilization efficiency
Please use more cautious wording here because nutrient availability and microbial utilization efficiency were not directly measured.
- Length of Discussion
The Discussion is quite lengthy and includes several mechanistic interpretations that were not directly evaluated in this study. Please consider condensing the Discussion and clearly distinguishing the observed results from proposed mechanisms.
Conclusion
- Biologically available substrates
Please use more cautious wording because nutrient accessibility was not directly measured in this study.
- Nutrient accessibility
Please use more cautious wording because nutrient accessibility was not directly measured in this study.
- Nutrient constraints and stoichiometric flexibility
This conclusion is too strong. Differences from global microbial stoichiometric ratios do not directly demonstrate nutrient constraints or microbial stoichiometric flexibility. Please revise this statement.
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AC2: 'Reply on RC2', Rabindra Adhikari, 05 Nov 2026
Response prepared for Referee #2 comments (egusphere-2026-3815):
“Soil horizon development and altitudinal-vegetational zonation decouple microbial biomass from bulk soil nutrient availability across a Himalayan treeline ecotone.”
(Note: for the clear distinction, the reviewer's comments are in light blue color and the corresponding responses are in automatic text colour.)
This manuscript investigates microbial biomass C, N, and P and microbial stoichiometry across soil horizons and altitudinal–vegetational zones in a Himalayan treeline ecotone. The study addresses an interesting topic and provides a useful dataset across contrasting vegetation zones and genetic soil horizons. However, several methodological and interpretative issues need further clarification. The main concerns relate to the preparation of soil samples before microbial biomass analysis, the calculation and interpretation of relative horizon contributions from mass-based concentrations, and several mechanistic interpretations that extend beyond the variables directly measured in this study. The statistical interpretation and consistency between the results and conclusions should also be carefully checked. My specific comments are provided below.
R: We thank the reviewer for the careful evaluation of our manuscript and for recognizing the value of the dataset across contrasting vegetation zones and soil horizons. We appreciate the constructive comments, which will help us improve the clarity, methodological transparency, and interpretation of our findings. In response, we will clarify the potential effects of soil sample preparation on microbial biomass measurements, revise the calculation and interpretation of horizon contributions, and ensure that our statistical results are described consistently with their significance levels. We will also revise the Discussion and Conclusion to distinguish the observed patterns in microbial biomass, soil nutrient concentrations, and microbial stoichiometry from potential mechanisms that were not directly assessed in this study. The specific comments are addressed individually below.
Abstract
Horizon contributions
The statement that the O-horizon accounted for 55.7%, 62.0%, and 62.8% of MBC, MBN, and MBP should be reconsidered. These values are based on mass-based concentrations rather than stocks. Concentrations from different horizons cannot be directly summed to represent their contribution to the whole soil profile without considering horizon thickness and soil mass or bulk density. Please revise this interpretation throughout the manuscript.
R: Thank you for this comment. We agree that the previously reported values of 55.7%, 62.0%, and 62.8% should not be interpreted as stocks. We will therefore remove these percentage-contribution estimates and the associated interpretation throughout in the revised manuscript.
The results will be presented in terms of microbial biomass concentrations for each soil horizon, and the interpretation will be revised accordingly. For example, we will describe the O-horizon as having the highest microbial biomass concentrations rather than assigning it a percentage contribution to the overall soil profile. This revision will be made consistently in the Abstract, Results, Discussion, and Conclusion to avoid interpreting horizon-level concentrations as profile-level biomass stocks.
Stoichiometric plasticity
The statement that the microbial biomass stoichiometry of 3.4:0.3:1 indicates stoichiometric plasticity is too strong. The observed ratios show differences from globally reported values, but they do not directly demonstrate microbial physiological plasticity. Please revise this interpretation.
R: Thank you for this valuable comment. We agree and therefore will revise the manuscript to distinguish the observed variation in microbial biomass stoichiometry from mechanistic interpretations. Statements implying that the observed ratio directly demonstrates stoichiometric plasticity or nutrient constraints will be revised. We will make these changes in the Discussion and Conclusion to ensure that our interpretations remain consistent with the variables measured in this study.
Introduction
Environmental stress
Please check the discussion of environmental stress. High pH is mentioned as an environmental stress, whereas the soils at the study site are described as strongly acidic. Please revise this paragraph to better reflect the conditions relevant to the study area.
R: We sincerely appreciate the reviewer’s careful observation. and will correct this statement and paragraph to refer to low pH in the revised manuscript to ensure consistency with the soil conditions at our study site.
Microbial CUE
Please use caution when discussing microbial CUE because CUE was not measured in this study. The relationship between the observed biomass stoichiometry and CUE should not be presented as a mechanism directly evaluated here.
R: Thank you for this important comment. Yes, it’s true that microbial carbon-use efficiency was not directly measured in this study. We will therefore revise this statement in the discussion section to avoid implying that the observed microbial biomass stoichiometry directly demonstrates changes in CUE. The discussion will now distinguish measured microbial biomass C:N ratios from “potential” mechanisms involving microbial carbon allocation and utilization.
Hypothesis 3
Please revise H3. Differences in microbial biomass stoichiometry can be tested directly, but “nutrient constraints” and “microbial adaptation” are mechanistic interpretations that were not directly measured in this study.
R: Thank you for this helpful suggestion. We agree and therefore will revise H3 to focus on measurable differences in microbial biomass C:N ratios among soil horizons and altitudinal–vegetational zones. This revision aligns the hypothesis with our measured variables and statistical analyses while avoiding assumptions about underlying mechanisms that were not directly evaluated. And we also consider the similar suggestion of Referee #1 to revise the H3 accordingly.
Materials and Methods
Soil sampling and laboratory analysis
Soil drying before microbial biomass analysis — major concern
Please justify the use of soil dried at 40°C for microbial biomass analysis. Drying followed by rewetting and three weeks of incubation may alter the microbial biomass compared with the original field condition. Please discuss how this sample preparation may affect the interpretation of MBC, MBN, and MBP.
R: Thank you for raising this important methodological concern. Soils were dried at 40°C, rewetted and pre-incubated for three weeks prior to analysis. MBC, MBN and MBP represent the microbial biomass re-established under standardized laboratory conditions rather than the in-situ field stock and should not be compared directly with absolute values measured on field moist soils. Drying rewetting is known to reduce microbial biomass, particularly MBP, and to alter community composition and elemental stoichiometry (Khan et al., 2019; Sawada et al., 2019; Sparling & Ross, 1988). Since all samples were treated identically, relative differences among sites are consistent and interpretable. They suit comparisons between treatments or sites that were all handled the same way.
Expression of soil properties
Relative contribution of soil horizons — major concern
Please reconsider the calculation and interpretation of the relative contribution of each horizon. Concentrations expressed per unit soil mass cannot be directly summed across horizons to represent their contribution to the whole soil profile without considering horizon thickness and soil mass or bulk density.
R: Thank you for highlighting this important calculation and interpretation of the result. As also addressed in our response to the reviewer’s earlier comment on horizon contributions in the Abstract, we agree that mass-based microbial biomass concentrations from different soil horizons should not be summed to estimate their relative contributions to the whole soil profile. The previously reported percentages were derived from horizon-specific concentration values and therefore should not have been interpreted as contributions to total soil-profile microbial biomass. We will take this into account throughout the text.
Statistical analysis
MBP model simplification
Please provide more details on the simplification of the MBP model. Specify which interaction terms were removed and how the final model structure was selected after the singularity problem was identified.
R: Thank you for this important comment. We agree that the statistical approach used for MBP requires further clarification. In our analysis, the mixed-effects model for MBP encountered a singularity issue. We therefore fitted an ordinary linear model, retaining Zone, Aspect, and Soil Horizon as fixed effects, together with all two-way interactions among these factors, but without the random intercept for Plot ID, which was used for MBC and MBN. Thus, the model structure was changed by excluding the random effect rather than by removing individual interaction terms. We will revise the Statistical Analysis section to clarify the model specification and distinguish the MBP analysis from the mixed-effects models used for MBC and MBN in our revised manuscript.
Zero or negative CFE flushes
Please provide further justification for treating all zero or negative CFE flushes as experimental artifacts and assigning them as missing values. Please clarify whether these observations were checked for analytical or procedural problems before exclusion.
R: Thank you for this important comment. We have checked the analytical records and calculations for the observations with zero or negative CFE flushes to assess potential analytical or procedural problems. As these non-positive net flushes do not provide valid positive estimates of microbial biomass using our calculation procedure, the affected observations were excluded from the corresponding biomass analyses and treated as missing values. We will revise the Methods section to clarify the checks performed, the rationale for exclusion, and the number of affected observations for MBC, MBN, and MBP. We will also acknowledge that these values may reflect very low microbial biomass or measurement variability and should not automatically be interpreted as confirmed experimental artifacts.
Results
Figures 3–6: Please improve the overall quality and readability of these figures. Some labels, symbols, and text are difficult to read at the current size. Please increase the figure resolution and ensure that all graphical elements remain clearly legible at publication size.
R: Thank you for this helpful suggestion. We will revise Figures 3–6 to improve their overall presentation and readability. We will increase the figure export resolution and adjusted text sizes, labels, symbols, and graphical layouts where necessary to ensure that the figures remain legible at publication size.
Soil organic carbon and total nitrogen
Profile contributions
Please revise this interpretation according to the comment in the Methods regarding the calculation of horizon contributions.
R: Thank you for this comment. As explained in our response to the earlier comments regarding horizon contributions, we have agreed that concentration-based measurements cannot be interpreted as contributions to the total soil profile. We will remove the profile contribution percentages and revise the corresponding interpretations of soil organic carbon (SOC) and total nitrogen (TN) throughout the manuscript, presenting the results in terms of horizon-specific concentrations instead.
Microbial biomass
Non-significant zone effect
The effect of altitudinal–vegetational zone was not significant (p = 0.259). Please avoid describing these differences as a distinct pattern and present them as descriptive trends only.
R: Thank you so much for pointing this out. We will revise the Results section to present the differences among zones as descriptive trends rather than statistically supported differences and ensure that the interpretation remains consistent with the statistical results.
p = 0.058
The interaction was not statistically significant at the stated α = 0.05 (p = 0.058). Please avoid referring to it as “marginally significant.”
R: Thank you so much for pointing out this. We will remove the term “marginally significant” and revise the Results section to report the interaction as statistically non-significant, avoiding interpretations that imply a statistically supported interaction.
Microbial stoichiometry
Overall, 3.4:0.3:1 ratio
Please clarify how the overall microbial biomass stoichiometry of 3.4:0.3:1 was calculated, particularly because MBC, MBN, and MBP contained different numbers of missing observations.
R: Thank you so much for this comment on how we calculated the tri-ratio. We have derived this tri-ratio by dividing the mean MBC and mean MBN setting MBP to 1.
Very high variability
The MBC:MBN ratio in the E-horizon shows extremely high variability (CV = 396.9%). Please discuss this variability when interpreting the approximately 17.8-fold difference from the O-horizon.
R: Thank you so much for this comment. We will revise the Results and Discussion to explicitly acknowledge the high within-horizon variability and clarify that the fold difference reflects a contrast in mean values rather than a uniform difference across samples.
Discussion
4.1 Vertical pedogenic control
“Biological activity”
Please revise “biological activity” because microbial biomass was measured, but microbial activity was not directly assessed.
R: Thank you so much for correctly pointing out this. Yes, we will revise the text and wording to refer to microbial biomass rather than activities so that the interpretation remains consistent with the measured variables.
4.2 Decoupling between microbial biomass and bulk soil nutrients
qMIC and microbial efficiency
This interpretation is too strong. Higher qMIC indicates a greater proportion of SOC present as microbial biomass, but it does not directly demonstrate more efficient substrate utilization or faster nutrient cycling.
Biologically available nutrient fractions
This statement is not directly supported by the measured variables. Biologically available nutrient fractions and microbial activity were not measured in this study. Please revise this interpretation.
R: Thank you for these comments regarding qMIC and biologically available nutrient fractions. We agreed and will revise the Discussion to ensure that our interpretations remain consistent. Specifically, we will clarify that qMIC indicates the proportion of organic carbon soil present as microbial biomass, rather than directly demonstrating more efficient substrate utilization. We will also rephrase the discussion of biologically available nutrient fractions to distinguish the observed patterns in microbial biomass and soil nutrient concentrations from interpretations involving nutrient availability. Thank you so much for this crucial comment.
4.3 Microbial stoichiometry and nutrient constraints
Stoichiometric plasticity
Please revise this interpretation according to the earlier comment regarding microbial stoichiometric plasticity.
R: Thank you for this comment. As addressed in our response to the earlier comment regarding stoichiometric plasticity, we will revise the Discussion to describe the observed differences in microbial biomass stoichiometry without interpreting deviations from globally reported ratios as direct evidence of microbial stoichiometric plasticity or physiological adaptation. We will not claim for direct evidence of microbial plasticity rather discuss deviation from global reported ratios.
Severe N limitation
Please use caution when interpreting high MBC:MBN ratios as evidence of severe N limitation. Nitrogen limitation and the proposed shifts toward maintenance and nutrient scavenging were not directly measured in this study.
R: We agreed and hence will revise the Discussion to present these mechanisms as possible explanations rather than demonstrated processes (e.g. MBC/MBN ratio as a relative amount of carbon and nitrogen in microbial biomass rather quantifying nitrogen availability) and to ensure that our interpretations remain consistent with the variables measured in this study.
4.4 Ecological implications
Nutrient availability and microbial utilization efficiency
Please use more cautious wording here because nutrient availability and microbial utilization efficiency were not directly measured.
R: Thank you so much for this crucial comment. We agreed and we will revise discussion section to distinguish our observed patterns in microbial biomass and soil nutrient concentrations from “potential” ecological explanations, avoiding conclusions about nutrient availability or microbial utilization efficiency.
Length of Discussion
The Discussion is quite lengthy and includes several mechanistic interpretations that were not directly evaluated in this study. Please consider condensing the Discussion and clearly distinguishing the observed results from proposed mechanisms.
R: Thank you for this helpful suggestion. We will condense the Discussion, particularly Sections 4.2–4.4, by removing repetitive content and reducing speculative mechanistic interpretations highlighted in our responses to the previous comments (e.g., explanations involving microbial utilizations efficiency, nutrient availability, and microbial stoichiometric plasticity). We will retain the key findings and focus on the measured variables, while revising or removing passages that are repetitive or extend beyond the evidence provided by our results.
Conclusion
Biologically available substrates
Please use more cautious wording because nutrient accessibility was not directly measured in this study.
Nutrient accessibility
Please use more cautious wording because nutrient accessibility was not directly measured in this study.
Nutrient constraints and stoichiometric flexibility
This conclusion is too strong. Differences from global microbial stoichiometric ratios do not directly demonstrate nutrient constraints or microbial stoichiometric flexibility. Please revise this statement.
R: Thank you for these comments. We will revise the Conclusion to use more cautious wording regarding nutrient accessibility, focused on directly measured variables in this study. Specifically, we will replace statements that imply differences in nutrient accessibility or substrate availability with descriptions of the observed patterns in microbial biomass and soil nutrient concentrations, presenting potential explanations as interpretations rather than direct findings. We will also revise statements concerning nutrient constraints and microbial stoichiometric flexibility. For example, rather than interpreting deviations from globally reported microbial stoichiometric ratios as direct evidence of nutrient constraints or microbial adaptation, we will describe the observed differences in microbial biomass C:N ratios and acknowledge that their underlying mechanisms cannot be established from our measurements alone. These revisions will ensure that the conclusions remain closely aligned with the results and the scope of the study.
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- 1
In this manuscript, Adhikari et al. investigated the spatial distribution and stoichiometric relationships of soil microbial biomass (MBC, MBN, MBP) across an altitudinal–vegetational gradient and four soil horizons in the Rolwaling Himalayan treeline ecotone, Nepal. This manuscript addresses a critical knowledge gap regarding soil microbial dynamics and nutrient stoichiometry in high-elevation ecosystems. The dataset combined both soil horizon differentiation and altitudinal-vegetational zonation in an understudied region (the Himalayas). A particularly interesting and well-presented finding is the empirical demonstration of a clear decoupling between bulk soil nutrient availability (SOC, TN) and microbial biomass (MBC, MBN), as well as the unusually low C:N:P ratio (3.4:0.3:1) indicating microbial stoichiometric plasticity under environmental stress. Overall, the manuscript is well-written and logically structured. However, there are several conceptual, methodological, and interpretive limitations that need to be addressed before publication. Below are my detailed major and minor comments: