Soil horizon development and altitudinal-vegetational zonation decouple microbial biomass from bulk soil nutrient availability across a Himalayan treeline ecotone

Adhikari, Rabindra; Huber, Jan; Maharjan, Amrit; Parajuli, Madhavi; Gall, Corinna; Seitz, Steffen; Subedi, Chandra Kanta; Schwab, Niels; Chaudhary, Ram Prasad; Schickhoff, Udo; Böhner, Jürgen; Oelmann, Yvonne; Scholten, Thomas

doi:10.5194/egusphere-2026-3815

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https://doi.org/10.5194/egusphere-2026-3815

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29 Jul 2026

| 29 Jul 2026

Status: this preprint is open for discussion and under review for SOIL (SOIL).

Soil horizon development and altitudinal-vegetational zonation decouple microbial biomass from bulk soil nutrient availability across a Himalayan treeline ecotone

Rabindra Adhikari, Jan Huber, Amrit Maharjan, Madhavi Parajuli, Corinna Gall, Steffen Seitz, Chandra Kanta Subedi, Niels Schwab, Ram Prasad Chaudhary, Udo Schickhoff, Jürgen Böhner, Yvonne Oelmann, and Thomas Scholten

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.

Received: 28 Jun 2026 – Discussion started: 29 Jul 2026

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RC1:
'Comment on egusphere-2026-3815', Anonymous Referee #1, 21 Sep 2026 reply
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:
line 48, please specify the altitude information (elevation range in meters above sea level, e.g., m a.s.l.) for the treeeline in the Rolwaling Himal to provide necessary spatial context for the reader.

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.

Lines 101–105: The main objective is not clearly described here. Please consider rephrasing this section.

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.

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.

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.

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).

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.

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.

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.

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.

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).

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.

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.

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.

Please consider increasing font size in figure 6 and figure 4.

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.

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.

Line 438, check the format of the unit “1300mm 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.

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.

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.

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.

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Citation: https://doi.org/10.5194/egusphere-2026-3815-RC1
RC2:
'Comment on egusphere-2026-3815', Anonymous Referee #2, 26 Sep 2026 reply
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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Citation: https://doi.org/10.5194/egusphere-2026-3815-RC2

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Short summary

Mountain treeline ecosystems are highly sensitive to climate change, yet the role of soil microbes remains poorly understood. We investigated microbial biomass and soil nutrients across a near-natural Himalayan treeline ecotone in Nepal. Our results show that soil horizon development and altitudinal-vegetation zonation are stronger controls on microbial biomass than bulk soil nutrient concentrations, providing new insights into nutrient cycling in mountain soils.


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