the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 15 May 2025
Fresh tephra deposits from the Tajogaite Volcano boost thermophile proliferation and soil organic matter recovery
Abstract. Tephra fallout deposition during volcanic eruptions overlays existing soils, profoundly altering their physical, chemical, and biological properties. This study investigates the impact of the newly deposited tephra blanket from the 2021 Tajogaite eruption (La Palma Island) on the molecular composition of soil organic matter and microbial diversity across different soil horizons. A combination of 16S and 18S rRNA gene sequencing, pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), and elemental and isotope ratio mass spectrometry (EA/IRMS) was employed. Our results demonstrate that tephra deposits significantly modify the organic matter composition of the underlying soils, promoting microbial activity linked to the degradation and transformation of organic carbon and nitrogen compounds. The soil horizon directly beneath the tephra layer (horizon O) displayed a higher abundance of labile organic compounds and a reduced presence of recalcitrant compounds compared to the deeper horizons (A and Bw). This pattern is strongly associated with the predominance of thermophilic bacteria, which contribute actively to the breakdown of complex organic materials such as lignin and hydrocarbons, and drive key biogeochemical processes including nitrogen and carbon cycling. The continuous geothermal influence of nearby fumaroles further supports the persistence and ecological success of thermophilic communities in these volcanic soils. These findings underscore the critical role of volcanic activity not only in reshaping soil structure but also in enhancing soil fertility and resilience through microbial-mediated processes. Understanding these dynamics is essential for soil management and ecosystem recovery strategies in volcanic regions, providing new insights into the long-term effects of tephra deposition on soil health and the carbon cycle.
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RC1: 'Comment on egusphere-2025-2086', Anonymous Referee #1, 05 Nov 2025
1 关键假设：引言最后陈述了研究目标，但缺乏明确的科学假设。这使得读者很难事先建立预期的逻辑框架。2 方法描述样本过多且无序。凋落物特性分析以及 DNA 提取、PCR 扩增和 Illumina MiSeq 测序样本过多。引物序列是什么？生物信息学分析和统计处理完全不同。本节不包含生物信息学分析。3 微生物样本的取样方法尚不清楚。是混合样本吗？包括重复多少次？该方法描述了 5 个样本的测定。但微生物学结果中的数据没有反映重复，没有生物统计学意义。4 根据论文规则，建议根据结果和讨论分章写。5 微生物剖面的结果过于简单化。建议增加对土壤元素和土壤微生物的分析。Citation: https://doi.org/
10.5194/egusphere-2025-2086-RC1 -
CC1: 'Reply on RC1', Nicasio T. Jiménez-Morillo, 28 Jan 2026
Dear Referee #1. Thank you very much for taking the time to read our article. We appreciate your inputs and comments. We hope the answers provided herein respond satisfactorily to your questions and recommendations. Please note that none of the authors speaks Chinese, which may affect the precision of our replies.
- Key Hypotheses: The introduction states the research objectives but lacks clear scientific hypotheses. This makes it difficult for readers to establish the expected logical framework beforehand.
Answer: Thank you for your comment. The last paragraph of the introduction has been modified to explicitly include the hypothesis as follows: “Under the hypothesis that tephra deposits from volcanic eruptions can modify the microbiota and the organic matter composition of the underlying soils, this work aimed to investigate the impact of the Tajogaite volcanic eruption on the molecular and microbial diversity of an existing soil profile covered by tephra deposits and elucidate its influence on soil resiliency and ecosystem recovery capacity. Using advanced molecular and microbiological techniques, including pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), stable isotope analysis, and 16S rRNA gene-sequencing, the soil profile was analysed to unravel how volcanic ash alters SOM quality and microbial community structure. ”
- Methods Description: Too many samples are described, and the descriptions of litter characterization, DNA extraction, PCR amplification, and Illumina MiSeq sequencing are excessive. What are the primer sequences? Bioinformatics analysis and statistical processing are completely different. This section does not include bioinformatics analysis.
Answer: We thank the reviewer for this helpful suggestion. We have thoroughly revised the Methods section to improve clarity and conciseness and to ensure that laboratory procedures, sequencing, bioinformatics, and downstream analyses are clearly separated:
Sample description and level of detail: We simplified the description of the amplicon dataset and reduced redundant methodological text. The revised section now focuses on the essential information required for reproducibility.
Primers and sequencing information: We added the complete primer information used to amplify the V3–V4 region, including primer names and sequences (341F: 5′-CCTACGGGNGGCWGCAG-3′; 805R: 5′-GACTACHVGGGTATCTAATCC-3′), and clarified that library preparation and sequencing (Illumina MiSeq, 2 × 250 bp) were performed by Novogene. This information is now provided in Section 2.4 (DNA extraction and 16S rRNA gene amplicon sequencing).
Bioinformatics vs statistical analyses: Following the reviewer’s recommendation, we separated bioinformatic processing into two well-differentiated subsections (Section 2.5, Bioinformatic processing and Section 2.6 Diversity and statistical analyses).
- 2.5. Bioinformatic processing:
Raw paired-end reads were processed using QIIME 2 (v2024.5) (Bolyen et al., 2019). Primer sequences were removed prior to denoising to prevent primer-derived artifacts and to improve read merging. Reads were then quality filtered and denoised using the q2-dada2 plugin (Callahan et al., 2016), which infers exact amplicon sequence variants (ASVs) by modeling sequencing errors and resolving unique biological sequences. Forward and reverse reads were merged to reconstruct the full V3–V4 amplicon, generating an ASV feature table for downstream ecological analyses. Chimeric sequences produced during PCR amplification were identified and removed using the consensus approach implemented in DADA2, reducing spurious diversity. Taxonomy was assigned with a Naïve Bayes classifier trained on the V3–V4 region against the SILVA reference database (release 138) (Quast et al., 2013), ensuring that taxonomic classification was optimized for the targeted amplicon region.
- 2.6. Diversity and statistical analyses
Alpha diversity analyse was computed in QIIME 2 (v2024.5). Alpha diversity was calculated to summarize within-sample diversity using richness and diversity indices (e.g., observed ASVs, Chao1, Shannon, and Simpson, as appropriate). To aid interpretation of the ordination, we produced a PCoA biplot by projecting (i) geochemical/pyrolytic descriptors (e.g., compound-class abundances from pyrolysis and elemental contents) and (ii) the relative abundances of dominant microbial phyla onto the same ordination space. Vectors (arrows) represent the direction of increasing values for each variable and their strength of association with the ordination axes (longer vectors indicate stronger relationships). This combined representation was used to highlight which chemical pools and microbial groups co-varied across samples and to identify the main gradients driving separation along PCo1 and PCo2.”
- Sampling Methods for Microbial Samples: Are they pooled samples? How many replicates are included? The method describes the determination of 5 samples. However, the data in the microbiological results do not reflect replication and lack biostatistical significance.
Answer: Thank you for your helpful comments. Each of the samples is composed of a mixture of 5 subsamples. Regarding the number of samples, there are 6 composed samples studied, to clarify this better in the article, the section “2.1 Study site and sampling” has been modified as follows:
- “Sampling included the collection of moss growing on the fresh tephra (MOSS), the tephra layer directly beneath the moss cover (moss substrate, MS), a dark green biofilm on horizon O (Biofilm), and three soil horizons: 0–2 cm (Horizon O) (0–2 cm), A (2–10 cm) (Horizon A) and Bw (10–20 cm) (Horizon Bw) (Fig. 1B). All samples analyzed in this study were composite samples, generated by collecting five subsamples per sample type across distinct sampling points and/or soil horizons, followed by thorough homogenization and pooling into a single representative mixture.”
- Regarding biostatistical significance, we analyzed five composite samples. Each composite was generated by pooling and thoroughly homogenizing five subsamples collected across different sampling points and/or horizons; therefore, each composite is intended to integrate within-site heterogeneity and provide a representative molecular and microbial profile of the corresponding condition. In addition, given the substantial analytical workload required for comprehensive molecular and microbial identification, together with the high cost of these analyses, this study was designed as an in-depth, high-resolution characterization. We consider this approach appropriate to obtain a robust and accurate overview of the dominant compositional patterns in these samples.
- According to the thesis guidelines, it is recommended to separate the results and discussion into chapters.
Answer: Thank you very much for your comment. We fully understand that separating the Results and Discussion sections can be beneficial. However, given the strongly multidisciplinary nature of the study and the use of advanced analytical techniques, we believe that presenting Results and Discussion together provides a clearer, more coherent view of the work and its objectives.
- Microbial profile results are overly simplified: It is recommended to add analyses of soil elements and soil microorganisms.
Answer: We greatly appreciate your comment. In response, we have added a new section presenting a comparative and statistical assessment of the analyzed samples based on principal coordinates analysis (PCoA), integrating elemental C and N data, the relative abundances of the main organic molecular families, and the dominant microbial taxa at the phylum level. This addition has substantially strengthened the manuscript by highlighting the relationships between molecular and microbial variables and clarifying their respective contributions and relevance across the different samples.
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AC3: 'Reply on RC1', Nicasio T Jiménez-Morillo, 10 Sep 2026
Dear Referee #1. Thank you very much for taking the time to read our article. We appreciate your inputs and comments. We hope the answers provided herein respond satisfactorily to your questions and recommendations. Please note that none of the authors speaks Chinese, which may affect the precision of our replies.
1.- Key Hypotheses: The introduction states the research objectives but lacks clear scientific hypotheses. This makes it difficult for readers to establish the expected logical framework beforehand.
Answer: Thank you for your comment. The last paragraph of the introduction has been modified to explicitly include the hypothesis as follows: “Under the hypothesis that tephra deposits from volcanic eruptions can modify the microbiota and the organic matter composition of the underlying soils, this work aimed to investigate the impact of the Tajogaite volcanic eruption on the molecular and microbial diversity of an existing soil profile covered by tephra deposits and elucidate its influence on soil resiliency and ecosystem recovery capacity. Using advanced molecular and microbiological techniques, including pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), stable isotope analysis, and 16S rRNA gene-sequencing, the soil profile was analysed to unravel how volcanic ash alters SOM quality and microbial community structure. ”
2.- Methods Description: Too many samples are described, and the descriptions of litter characterization, DNA extraction, PCR amplification, and Illumina MiSeq sequencing are excessive. What are the primer sequences? Bioinformatics analysis and statistical processing are completely different. This section does not include bioinformatics analysis.
Answer: We thank the reviewer for this helpful suggestion. We have thoroughly revised the Methods section to improve clarity and conciseness and to ensure that laboratory procedures, sequencing, bioinformatics, and downstream analyses are clearly separated:
Sample description and level of detail: We simplified the description of the amplicon dataset and reduced redundant methodological text. The revised section now focuses on the essential information required for reproducibility.
Primers and sequencing information: We added the complete primer information used to amplify the V3–V4 region, including primer names and sequences (341F: 5′-CCTACGGGNGGCWGCAG-3′; 805R: 5′-GACTACHVGGGTATCTAATCC-3′), and clarified that library preparation and sequencing (Illumina MiSeq, 2 × 250 bp) were performed by Novogene. This information is now provided in Section 2.4 (DNA extraction and 16S rRNA gene amplicon sequencing).
Bioinformatics vs statistical analyses: Following the reviewer’s recommendation, we separated bioinformatic processing into two well-differentiated subsections (Section 2.5, Bioinformatic processing and Section 2.6 Diversity and statistical analyses).
- 2.5. Bioinformatic processing:
Raw paired-end reads were processed using QIIME 2 (v2024.5) (Bolyen et al., 2019). Primer sequences were removed prior to denoising to prevent primer-derived artifacts and to improve read merging. Reads were then quality filtered and denoised using the q2-dada2 plugin (Callahan et al., 2016), which infers exact amplicon sequence variants (ASVs) by modeling sequencing errors and resolving unique biological sequences. Forward and reverse reads were merged to reconstruct the full V3–V4 amplicon, generating an ASV feature table for downstream ecological analyses. Chimeric sequences produced during PCR amplification were identified and removed using the consensus approach implemented in DADA2, reducing spurious diversity. Taxonomy was assigned with a Naïve Bayes classifier trained on the V3–V4 region against the SILVA reference database (release 138) (Quast et al., 2013), ensuring that taxonomic classification was optimized for the targeted amplicon region.
- 2.6. Diversity and statistical analyses
Alpha diversity analyse was computed in QIIME 2 (v2024.5). Alpha diversity was calculated to summarize within-sample diversity using richness and diversity indices (e.g., observed ASVs, Chao1, Shannon, and Simpson, as appropriate). To aid interpretation of the ordination, we produced a PCoA biplot by projecting (i) geochemical/pyrolytic descriptors (e.g., compound-class abundances from pyrolysis and elemental contents) and (ii) the relative abundances of dominant microbial phyla onto the same ordination space. Vectors (arrows) represent the direction of increasing values for each variable and their strength of association with the ordination axes (longer vectors indicate stronger relationships). This combined representation was used to highlight which chemical pools and microbial groups co-varied across samples and to identify the main gradients driving separation along PCo1 and PCo2.”
3.- Sampling Methods for Microbial Samples: Are they pooled samples? How many replicates are included? The method describes the determination of 5 samples. However, the data in the microbiological results do not reflect replication and lack biostatistical significance.
Answer: Thank you for your helpful comments. Each of the samples is composed of a mixture of 5 subsamples. Regarding the number of samples, there are 6 composed samples studied, to clarify this better in the article, the section “2.1 Study site and sampling” has been modified as follows:
- “Sampling included the collection of moss growing on the fresh tephra (MOSS), the tephra layer directly beneath the moss cover (moss substrate, MS), a dark green biofilm on horizon O (Biofilm), and three soil horizons: 0–2 cm (Horizon O) (0–2 cm), A (2–10 cm) (Horizon A) and Bw (10–20 cm) (Horizon Bw) (Fig. 1B). All samples analyzed in this study were composite samples, generated by collecting five subsamples per sample type across distinct sampling points and/or soil horizons, followed by thorough homogenization and pooling into a single representative mixture.”
- Regarding biostatistical significance, we analyzed five composite samples. Each composite was generated by pooling and thoroughly homogenizing five subsamples collected across different sampling points and/or horizons; therefore, each composite is intended to integrate within-site heterogeneity and provide a representative molecular and microbial profile of the corresponding condition. In addition, given the substantial analytical workload required for comprehensive molecular and microbial identification, together with the high cost of these analyses, this study was designed as an in-depth, high-resolution characterization. We consider this approach appropriate to obtain a robust and accurate overview of the dominant compositional patterns in these samples.
4.- According to the thesis guidelines, it is recommended to separate the results and discussion into chapters.
Answer: Thank you very much for your comment. We fully understand that separating the Results and Discussion sections can be beneficial. However, given the strongly multidisciplinary nature of the study and the use of advanced analytical techniques, we believe that presenting Results and Discussion together provides a clearer, more coherent view of the work and its objectives.
5.- Microbial profile results are overly simplified: It is recommended to add analyses of soil elements and soil microorganisms.
Answer: We greatly appreciate your comment. In response, we have added a new section presenting a comparative and statistical assessment of the analyzed samples based on principal coordinates analysis (PCoA), integrating elemental C and N data, the relative abundances of the main organic molecular families, and the dominant microbial taxa at the phylum level. This addition has substantially strengthened the manuscript by highlighting the relationships between molecular and microbial variables and clarifying their respective contributions and relevance across the different samples.
Citation: https://doi.org/10.5194/egusphere-2025-2086-AC3
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CC1: 'Reply on RC1', Nicasio T. Jiménez-Morillo, 28 Jan 2026
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CC2: 'Comment on egusphere-2025-2086', Layla Márquez San Emeterio, 20 Feb 2026
This study is of great interest due to its relevance, especially considering the magnitude of the recent geological event in the Canary Islands, the complexity of the experimental approach, and the significance of the findings. However, some improvements and additional information are needed, particularly in the discussion section. In addition, the manuscript would benefit from clearly stated hypotheses. In general terms, I recommend its publication after these major revisions (see pdf file attached with some comments).
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AC1: 'Reply on CC2', Nicasio T Jiménez-Morillo, 18 May 2026
We sincerely thank you for carefully reviewing our manuscript and for providing valuable comments and suggestions. We appreciate the time and effort devoted to evaluating our work, and we hope that the responses provided below adequately address your questions and recommendations.
Comment 1
“lack of novelty, I believe this is the first study in this area combining these analytical approaches, also given the relevance of this event when it happened”
Response:
We thank the reviewer for this comment. We agree that the novelty of the study was not sufficiently emphasized in the Abstract. To address this, we have revised the Abstract to explicitly highlight the novelty of our integrative analytical approach combining Py-GC/MS, stable isotope analysis, and microbial sequencing to investigate the early transformation of soils affected by the Tajogaite eruption.Revised text:
Lines 19-20: “To our knowledge, this is the first study combining molecular pyrolysis, stable isotope analysis, and microbial sequencing to characterize the early biogeochemical transformation of soils affected by the Tajogaite eruption.”Comment 2
“7,000”
Response:
We thank the reviewer for this correction. The number of displaced residents has been updated to include the appropriate thousands separator for clarity.Revised text:
Line 43: “Over 85 days, the eruption released over 200 million cubic meters of lava and pyroclasts, burying over 1,200 hectares of land and displacing 7,000 residents.”Comment 3
“I would be convenient to define what's lapilli in advance”
Response:
We thank the reviewer for this helpful suggestion. The sentence has been modified to clearly define the grain size range of lapilli and improve clarity.Revised text:
Lines 46-47: “According to these authors, the grain size of the tephra blanket varied during the eruption from lapilli (2–64 mm) to very fine ash (<2 mm) that spread to La Gomera, El Hierro, Tenerife and Gran Canaria islands during the more explosive phases.”Comment 4
“I think these statements would make more sense after the sentence in which you start describing the changes in soil fertility, etc.”
Response:
We thank the reviewer for this suggestion. The sentence has been relocated to the paragraph describing the effects of tephra deposition on soil fertility and soil properties in order to improve the logical flow of the introduction.Revised text:
Lines 61-64: Ashfall from Tajogaite eruption has been reported to significantly impact agricultural productivity (Sánchez-España et al., 2023; Taddeucci et al., 2023). The loss of fertile topsoil and the alteration of soil properties created long-term challenges for agricultural recovery and land use (Ustiatik et al., 2023).Comment 5
“feels like this is redundant with the information given above”
Response:
We agree with the reviewer that this sentence was redundant with the previous paragraph. To improve clarity and avoid repetition, the redundant sentence has been removed and the information has been integrated into a revised paragraph describing the effects of tephra deposition on soil properties and ecosystem recovery.Comment 6
“same as pointed out above for nutrient retention, I may suggest to keep a structure for describing the effect of tephra on both factors”
Response:
We thank the reviewer for this insightful suggestion. Following this recommendation, the three paragraphs discussing the effects of tephra on soil properties, nutrient dynamics, and microbial communities have been reorganized and merged into a single coherent section to avoid redundancy while preserving all references.Revised text:
Lines 52-64: Furthermore, the interactions between volcanic ashfall and soil organic matter (SOM) can lead to the formation of polycyclic aromatic hydrocarbons (PAHs) and other condensed compounds (Nanzyo et al., 1993; Tomašek et al., 2021). These processes can influence soil fertility, stability, and overall health by modifying nutrient availability, pH, and soil aggregation (Arias et al., 2005; Peng et al., 2021; De la Rosa et al., 2023). Additionally, volcanic ash can promote the formation of new organo-mineral complexes, altering SOM decomposition pathways and the long-term sequestration of carbon and other essential nutrients (Nierop and Buurman, 2007; Hernández et al., 2012; Iwasaki et al., 2021) to better support plant and microbial communities, ultimately influencing the trajectory of ecosystem recovery (Hernández et al., 2012; Yokobe et al., 2020; Muñoz et al., 2021). Therefore, tephra deposition can also reshape soil microbial communities by creating new ecological niches that favour specific microbial taxa adapted to volcanic substrates (Zeglin et al., 2016; Yokobe et al., 2020; Chen et al., 2021). Understanding these processes is crucial, particularly in volcanic regions where soil resilience is key to ecosystem recovery because ashfall from Tajogaite eruption has been reported to significantly impact agricultural productivity (Sánchez-España et al., 2023; Taddeucci et al., 2023). The loss of fertile topsoil and the alteration of soil properties created long-term challenges for agricultural recovery and land use (Ustiatik et al., 2023).”Comment 7
“What's the knowledge gap of your study? I am also missing the hypotheses beyond the main objective of your study”
Response:
We thank the reviewer for this important comment. The final paragraph of the Introduction has been rewritten to more clearly identify the knowledge gap addressed by this study and to better articulate the objective of the work.Revised text:
Lines 67-76: Despite increasing interest in the ecological consequences of volcanic eruptions, the combined effects of fresh tephra deposition on SOM molecular composition and microbial community structure remain poorly understood, particularly during the early stages of soil recovery following volcanic disturbance. This study aims to investigate how the tephra blanket produced by the 2021 Tajogaite eruption has altered the molecular composition of SOM and the structure of microbial communities in a pre-existing soil profile. By integrating analytical pyrolysis (Py-GC/MS), stable isotope analysis, and 16S and 18S rRNA gene sequencing, we evaluate the interactions between newly deposited volcanic materials, organic matter transformation, and microbial colonization. Understanding these processes may provide new insights into the mechanisms driving early soil regeneration and ecosystem recovery in recently disturbed volcanic environments.Comment 8
“General comments: I suggest creating two different subparargaphs, one for describing the study site and other for the sampling for soil and pyroclastic material, since the site itself is quite complex”
Response:
We thank the reviewer for this very helpful suggestion. Following this recommendation, the section has been reorganized into two separate subsections to improve clarity: one describing the study site and another describing the sampling procedures.Revised structure:
2.1 Study site
Text describing the geological setting and environmental characteristics of the sampling area (from “The soil profile studied is located in the Las Manchas area…” to “…temperatures higher than the ambient (Campeny et al., 2023; Martínez-Martínez et al., 2023)”).2.2 Sampling
Text describing the sampling procedures (from “All samples were collected using…” to “…every hour for one year.”).Comment 9
“has this been described elsewhere?”
Response:
We thank the reviewer for this comment. The geological evolution of La Palma has been extensively described in previous studies. To clarify this point, we have added the appropriate references supporting this statement.Revised text:
Line 85: (Carracedo et al., 2001; Sánchez-España et al., 2023).Comment 10
“this might belong to the introduction part, although you have mentioned that already”
Response:
We agree with the reviewer that this sentence is redundant with the information already presented in the Introduction. Therefore, the sentence has been removed from the manuscript to improve conciseness.The removed sentence was:
“This site provides a unique opportunity to study the interactions between fresh tephra deposits and the underlying soil, particularly their effects on soil structure, organic matter composition, and microbial communities.”
Comment 11
“this should be described above (aprox. line 102) when you mention the fumarole gases”
Response:
We thank the reviewer for this helpful suggestion. Following this recommendation, the description of the environmental monitoring system has been relocated to improve the logical flow of the Methods section (lines 109-111).Comment 12
“you should mention at least what you used for this decarbonization and for how long, even though you keep those citations”
Response:
We thank the reviewer for this suggestion. To improve clarity and reproducibility, we have added a brief description of the decarbonization procedure used prior to isotopic analysis following the protocols described in Jiménez-Morillo et al. (2016, 2020a).Revised text:
Lines 125-127: Briefly, samples were treated with 10% HCl for 24 hours to eliminate carbonate phases and subsequently rinsed with deionized water until a neutral pH was reached. The treated samples were then dried at 65 °C before isotopic analysis.Comment 13
“use ‘/’ instead of ‘|’ to indicate the isotope ratios”
Response:
We thank the reviewer for this suggestion. However, the notation using the slash “/” is the most widely used convention in isotope geochemistry to indicate isotope ratios (e.g., δ¹³C and δ¹⁵N expressed relative to reference standards). Therefore, we have maintained the commonly used notation throughout the manuscript to ensure consistency with standard reporting practices.Comment 14
“which standards?”
Response:
We thank the reviewer for pointing this out. The standards used for isotope calibration have now been specified in the manuscript to improve methodological clarity.Revised text:
Lines 131-134: Isotope values are expressed in delta (δ) notation in parts per thousand (‰) relative to certified standards set by the International Atomic Energy Agency (IAEA). For 𝛿13C, the standards were: cellulose (IAEA-CH-3, 𝛿13C = -24.7‰); sucrose (IAEA-CH-6, 𝛿13C = -10.5‰), and caffeine (IAEA-600, 𝛿13C = -27.8‰), while for 𝛿15N, the standards were: caffeine (IAEA-600, 𝛿15N = 1.0‰); ammonium sulfate (IAEA-N1, 𝛿15N = 0.4‰), and ammonium sulfate (IAEA-N2, 𝛿15N = 20.4‰). with a standard deviation for bulk δ13C of ± 0.05 ‰ and δ15N of ± 0.1 ‰. Duplicate analyses were performed for samples and standards (n = 2).
Comment 15
“split ratio?”
Response:
We thank the reviewer for this comment. However, in this study, samples were introduced using splitless injection mode; therefore, no split ratio was applied.Comment 16
“but why have you chosen those categories if that study is based on soil samples after a forest fire? does this classification apply as well to the pyroclastic material (not soils)? please explain further”
Response:
We thank the reviewer for this comment. This classification was selected because it encompasses the main organic families commonly identified in both thermally altered soils, such as fire-affected soils, and unburned control soils. Moreover, this approach is consistent with a substantial body of published literature that applies the same classification framework, including studies not specifically focused on burned soils.Comment 17
“what about statistical analyses?”
Response:
We thank the reviewer for this comment. To improve the description of the analytical procedures, a new subsection describing the statistical analyses performed in this study has been added to the Methods section.Revised text (new subsection):
Lines 172-185:
2.7 Diversity and statistical analyses
All analytical measurements were performed in duplicate and results are reported as mean values with their corresponding standard deviations. Statistical differences among samples were evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test at a significance level of p < 0.05. These analyses were used to identify statistically significant differences in elemental and isotopic composition among the different soil horizons, biofilm samples, and moss substrates. Correlation analyses between δ¹³C and carbon content were performed using linear regression in order to evaluate relationships between isotopic composition and organic carbon abundance. Alpha diversity analyse was computed in QIIME 2 (v2024.5). Alpha diversity was calculated to summarize within-sample diversity using richness and diversity indices (e.g., observed ASVs, Chao1, Shannon, and Simpson, as appropriate). To aid interpretation of the ordination, we produced a PCoA biplot by projecting (i) geochemical/pyrolytic descriptors (e.g., compound-class abundances from pyrolysis and elemental contents) and (ii) the relative abundances of dominant microbial phyla onto the same ordination space. Vectors (arrows) represent the direction of increasing values for each variable and their strength of association with the ordination axes (longer vectors indicate stronger relationships). This combined representation was used to highlight which chemical pools and microbial groups co-varied across samples and to identify the main gradients driving separation along PCo1 and PCo2.
Comment 18
“if this refers to organic C, you should refer it as TOC”
Response:
We thank the reviewer for this suggestion. The terminology has been corrected throughout the manuscript and in Table 1. The column previously labelled “C (%)” has been replaced by “TOC (%)” to clearly indicate that the values correspond to total organic carbon.Revised text:
Line 190 and thereafter: In Table 1 and throughout the manuscript, C (%) has been replaced by TOC (%) to indicate total organic carbon.Comment 19
“you're merely describing the data that I can already check on the table, I may suggest to describe the magnitude of the differences (i.e., horizon O resulted in ca. X times higher the C content compared to…)”
Response:
We thank the reviewer for this suggestion. Following this recommendation, the text has been revised to better emphasize the magnitude of the differences among samples rather than simply repeating the numerical values shown in Table 1.Revised text:
“Horizon O samples exhibited the highest average of TOC (between 1.4 and 6.4 fold), which may be associated with the presence of a noticeable root layer (Fig. 1). The content of N was also the highest, although no significant difference was found with the Biofilm samples collected from Horiazon O. The biofilm also shows notable TOC, and its ratio TOC/N close to 9 (8.83 on average) indicates that the biofilm was found in a healthy state.
Comment 20
“References?”
Response:
We thank the reviewer for this comment. The interpretation provided in this section is primarily based on direct observations of the sampled material, which showed a high abundance of roots within the MS layer in addition to tephra particles. To clarify the possible contribution of roots to the organic carbon and nitrogen content observed in this material, we have added references describing the role of root exudates as an important source of carbon and nitrogen in soils.Revised text:
Lines 208-209: Additionally, the presence of root exudates, rich in C and N compounds, may contribute to this remarkable concentration of organic compounds (Sokolova, 2020).
Comment 21
“are any of these colonizers producing mycorrhizaes you attributed before?”
Response:
We thank the reviewer for this interesting question. In this study, the presence of early colonizing microorganisms was inferred from the microbial community analysis (16S rRNA) and previous studies describing biological colonization in recently disturbed volcanic environments. However, the presence of mycorrhizae was neither directly assessed nor mentioned in this work.Comment 22
“same as commented for C and N contents: I can already check the values as depicted in the table, but you should aim at pointing out the magnitude of the differences comparing the different samples”
Response:
We thank the reviewer for this suggestion. Following this recommendation, the text has been modified to highlight the magnitude of the isotopic differences among the different samples.Revised text:
“The observed ¹³C-enrichment of the MS (about 4–5 ‰ above Moss, Biofilm and Horizon O samples), may be attributed to…”
Comment 23
“are”
Response:
We thank the reviewer for pointing out this wording issue. However, in this case the term used in the manuscript correctly refers to the composition of the MS layer, which is mainly composed of tephra. Therefore, the original wording has been maintained.Comment 24
“I feel that the presence of this material is not entirely discussed or justified, could you improve that?”
Response:
We thank the reviewer for this comment. To avoid possible confusion regarding the terminology used, we have clarified the text by referring to tephra rather than lapilli, which is a specific grain-size fraction of tephra deposits.Revised text:
Lines 225-228: This could be indicative of the volcanic origins of the paleo soil (Horizons A and B) (Carracedo et al., 2022) and the importance of tephra, among other volcanic materials from previous eruptions, in the carbon cycle during soil formation (Matus et al., 2014 and references therein).
Comment 25
“superscript”
Response:
We thank the reviewer for pointing out this formatting issue. The notation in Figure 2 has been corrected and the ¹³C isotope is now presented in superscript in the y-axis label.Comment 26
“indicate units in the axis titles”
Response:
We thank the reviewer for this comment. The units have now been added to the y-axis label in Figure 2 to improve clarity.Revised text:
“The y-axis label in Figure 2 has been updated to include the unit ‰.”
Comment 27
“so is this somehow a limitation from your study? any message that should be taken from this observation? please discuss any further”
Response:
We thank the reviewer for this insightful comment. This observation does not represent a limitation of the present study but rather reflects the different origin and developmental stage of the analysed materials. In particular, the moss and MS samples were collected directly from the recently deposited tephra layer, whereas the other samples correspond to the underlying soil horizons (O, A and Bw). As a result, these materials belong to different stages of soil development, which explains the lack of correlation observed between the tephra-derived samples and the pre-existing soil horizons. To clarify this point, we have expanded the discussion in the revised manuscriptRevised text:
Lines 244-248: This is probably because the samples were collected shortly after the volcanic eruption that accumulated the tephra over the previous soil (Horizon O). Indicating that the C cycle of the tephra layer was still in an early stage of the andisol formation process, preventing the observation of statistical correlations between the soil samples and the tephra. Future analyses of the same tephra layer could better constrain the process of andisol formation as it gets closer to the regression line of horizons depicted in figure 2.
Comment 28
“I dont like the use of 'exhibited' for describing the results; you can simply use 'increase', 'decrease', 'observed'.”
Response:
We thank the reviewer for this suggestion. The wording has been revised to avoid the use of “exhibited” and to use a more direct description of the observed results.Revised text:
249-250: Regarding the nitrogen isotope composition (δ¹⁵N), the moss sample showed the lowest δ¹⁵N value (Table 1), a typical characteristic for mosses (Zechmeister et al., 2008).
Comment 29
“the reader may be lost at this point because what's the relationship between the composition of bryophytes and your samples? please explain further.”
Response:
We thank the reviewer for this comment. We agree that the reference to bryophytes was introduced without sufficient context. To improve clarity, we have revised the text to better explain the relationship between the composition of bryophytes and the analysed samples.Revised text:
Lines 266-267: Polysaccharides are commonly associated with bryophytes such as mosses, serving as structural components and also assumed to contribute to stress tolerance (Klavina et al., 2015).
Comment 30 (lines 237–238)
“so are these biomarkers suitable for characterizing these samples or not?”
Response:
We thank the reviewer for this important question. The use of analytical pyrolysis have demonstrated to be suitable for a wide range of organic compounds. The sentence referred to describes the limitations of the technique with regard to the complete identification of the polysaccharides. The technique is able to identify all the polysaccharides described in the manuscript, but we warn the reader that other polysaccharides may also be present in the samples analysed. We have clarified this point in the manuscript.Revised text:
Line 269-270: However, due to dehydration and molecular restructuring during pyrolysis, complete identification of polysaccharides is not feasible, and other polysaccharides may be present (González-Pérez et al., 2016)
Comment 31 (line 246)
“why is important or relevant to refer to this pathogen thing?”
Response:
We thank the reviewer for this comment. The reference to plant defence mechanisms was included to explain the origin of certain bioactive compounds detected in the samples. To clarify this point, we have slightly revised the text to better connect the production of these compounds with plant defence strategies.Revised text:
“Plants produce a wide range of bioactive metabolites, peptides, and small molecules that contribute to defence mechanisms and microbial resistance (Valeeva et al., 2022), which may explain the presence of some of the compounds detected in the Moss samples.”Comment 32 (general comment below line 256)
“Discussion is well supported by references but I feel there is lack of connection among all the statements, I suggest making a smoother narrative for discussing and justifying your results, rather than merely describing the data and adding references without linking it with your hypothesis.”
Response:
We thank the reviewer for this valuable suggestion. Following this recommendation, the first part of Section 3.2 has been revised to improve the narrative flow of the discussion and to better connect the interpretation of the results with the objectives and hypotheses of the study.Comment 33 (general comment below line 281)
“the discussion here is better linked with the results, but I suggest linking it with your hypotheses.”
Response:
We thank the reviewer for this suggestion. The discussion has been revised to more explicitly relate the interpretation of the results with the hypotheses proposed in the Introduction.Comment 34 (lines 377–379)
“why is this noteworthy?”
Response:
We thank the reviewer for this comment. We agree that the relevance of this observation was not sufficiently explained. The text has been revised to clarify why the high microbial diversity detected in the tephra layer is remarkable considering the recent volcanic disturbance.Revised text:
Lines 401-412: It is noteworthy that the microbial community in the tephra collected beneath the moss (MS) reached a substantial diversity and richness within just two years following the deposition of the Tajogaite volcanic tephra, because it suggests a rapid microbial colonization of the volcanic substrate
Comment 35 (lines 391–392)
“do you think you can link these findings with your biomarkers data retrieved by Py-GC/MS?”
Response:
We thank the reviewer for this valuable suggestion. In response, and in line with the comments raised by Reviewer 1, we performed a statistical analysis (PCoA) to examine the relationships between the main geochemical variables, including elemental composition and the relative abundance of the major organic families, and the most abundant microbial phyla. These results are now presented in Section 3.5, entitled “Linkages between biogeochemical data and microbial community composition.” This new analysis revealed significant correlations between the molecular data and microbial community composition, supporting the existence of clear links between biogeochemical characteristics and microbial assemblages. In particular, the results allowed the identification of biomarkers, or groups of biomarkers, associated with soil alteration caused by volcanic ash deposition, as well as with moss development on the surface of the ash layer.Comment 36 (lines 414–415)
“confirmed how? please be more specific.”
Response:
We thank the reviewer for this suggestion. The text has been removed to avoid confusion, because, we could not analyse the elemental composition of sulphur in the samples.Comment 37 (lines 461–474)
“I think this paragraph describing general findings should either be placed at the beginning of this section, or rewrite it as a wrap-up paragraph, otherwise it seems that you're repeating the same information.”
Response:
We thank the reviewer for this helpful suggestion. Following this recommendation, the paragraph has been revised and repositioned to function as a wrap-up paragraph at the end of the section. In its revised form, it now provides a concise synthesis of the main findings and links the microbial results with their broader ecological implications for volcanic soils, thereby avoiding repetition of information already presented above.Revised text:
Lines 497-503 “Thermophilic and thermotolerant bacteria constitute one of the most ecologically relevant features of this tephra-soil system and provide a strong biological signal of the persistent geothermal influence affecting the sampled profile. Their marked abundance in horizons O and Bw, as well as in the biofilm, strongly suggests that residual heat and fumarolic activity associated with the Tajogaite volcanic system continue to shape microbial community assembly two years after the eruption. This interpretation is particularly meaningful because thermophilic lineages were detected across multiple compartments of the profile rather than being restricted to a single layer, indicating that thermal disturbance has influenced both surface-associated and buried soil communities (Picone et al., 2020; Hernández et al., 2020).”Comment 38 (lines 475–485)
“same for previous paragraph, i think thermophiles are of a great interest for your study but its discussion feels a bit weak.”
Response:
We thank the reviewer for this valuable comment. We agree that thermophilic microorganisms are of particular relevance in this study. Accordingly, this part of the discussion has been expanded and strengthened to better explain their ecological significance in the tephra-soil system, with particular emphasis on their association with geothermal influence, their potential role in the transformation of complex organic matter, and their contribution to early soil development after tephra deposition.Revised text:
Lines 504-520 “Several of the genera identified in these samples, including Thermasporomyces, Thermopolyspora, Thermobacillus, Thermoleophilum, Sphaerobacter, Thermogemmatispora and Litorilinea, are thermophilic bacteria known to participate in the degradation of lignocellulosic substrates, hydrocarbons and other structurally complex organic compounds. Their distribution is therefore consistent with the molecular patterns identified by Py-GC/MS (Fig.3). In particular, the presence of lignin-derived compounds in Horizon O and the occurrence of PAHs and related aromatic compounds across the soil profile suggest that these microorganisms may be actively involved in the turnover and transformation of organic matter under geothermal influence. In this context, thermophiles should not be viewed merely as passive indicators of elevated temperature, but as potential drivers of biogeochemical processing during the early stages of soil recovery after tephra deposition (Nzila, 2018).Their ecological relevance is further supported by the observed coexistence of thermophilic (e.g., Thermasporomyces, Thermopolyspora, Thermobacillus) and non-thermophilic (e.g., Acidobacter, Norcadioides, Planifium) taxa, which points to a dynamic microbial system structured by fluctuating thermal conditions rather than by permanently extreme temperatures. Such variability is consistent with the environmental monitoring data, which showed notable temporal oscillations in temperature and humidity at the sampling site. This combination of thermal heterogeneity, newly deposited volcanic material, and fresh organic inputs from mosses and biofilms likely creates a mosaic of ecological niches that favours functionally diverse microbial assemblages. Altogether, these results support the view that geothermal influence not only selects for specialized microbial taxa, but may also accelerate organic matter transformation, nutrient recycling and the first stages of andisol development in this recently disturbed volcanic environment (Guo et al., 2014; Hernández et al., 2020).”
Comment 39 (Conclusions section)
“conclusions are mostly comprising a repetition of results and discussion, you should shorten this part and refer to the main findings, as well as highlighting the relevance and novelty of your study. also, any limitations or further discussion/research that should be conducted?”
Response:
We thank the reviewer for this valuable suggestion. The Conclusions section has been revised and shortened to focus on the main findings of the study while highlighting its novelty and relevance. We have also added a brief statement discussing future research directions to better understand the long-term evolution of volcanic soils after tephra deposition.Revised text:
Lines 574-596:
- Conclusions
¨This study provides new insights into the early stages of soil development following the deposition of volcanic tephra produced by the 2021 Tajogaite eruption (La Palma Island). The results show that moss colonizing the upper tephra layer contributes organic compounds that serve as carbon sources for microorganisms, promoting rapid microbial colonization of the recently deposited volcanic substrate within only two years after the eruption.
Molecular analyses revealed clear differences between the organic matter composition of the tephra layer and the underlying soil horizons. Horizon O displayed a distinctive hydrocarbon signature associated with both thermal alteration processes and active microbial transformation of organic matter, whereas the deeper soil horizons (A and Bw) retained alkane patterns characteristic of plant-derived inputs. The δ¹³C isotopic signatures further support intense microbial processing of organic carbon in the upper soil horizon, indicating that microbial activity plays a key role in the transformation of organic matter shortly after tephra deposition.
Microbial sequencing revealed the presence of Cyanobacteriota in the biofilm and surface-associated samples, highlighting the role of photosynthetic microorganisms as early colonizers contributing to primary production, nitrogen input, and substrate stabilization. The presence of ammonia-oxidizing archaea, sulfur-oxidizing bacteria, methane-utilizing microorganisms, ferric iron-reducing taxa, and hydrocarbon-metabolizing microorganisms, including members of the family Methyloligellaceae, indicates that key biogeochemical cycles were already active only two years after tephra deposition.. In addition, the widespread presence of thermophilic microorganisms across the soil profile highlights the influence of geothermal activity associated with fumaroles in shaping microbial community composition and promoting the degradation of complex organic compounds in these recently disturbed soils.
Overall, our results demonstrate that fresh tephra deposition can strongly influence soil organic matter dynamics and microbial community structure during the early stages of volcanic soil formation. These findings highlight the important role of thermophilic microorganisms and pioneer vegetation in driving the initial development of andisols and the recovery of soil ecosystems following volcanic disturbances.
To our knowledge, this is the first study integrating molecular pyrolysis, isotopic analysis and microbial sequencing to characterize the early biogeochemical transformation of tephra deposits after the Tajogaite eruption. Future studies monitoring the same tephra deposits over time will be essential to better understand the long-term evolution of organic matter transformation and microbial succession during andisol formation in volcanic environments¨.
Citation: https://doi.org/10.5194/egusphere-2025-2086-AC1
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AC1: 'Reply on CC2', Nicasio T Jiménez-Morillo, 18 May 2026
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RC2: 'Comment on egusphere-2025-2086', Anonymous Referee #2, 18 Aug 2026
Opening note
Overall, I think the synthesized dataset (combining Py-GC/MS, isotopes, and 16S) is a genuinely interesting and fairly rich resource, and I find the core observation that tephra deposition alters SOM composition and microbial activity plausible. However, I am concerned about how far the interpretation is pushed beyond what a single-site, low-replication, comparison-free design can support. Furthermore, I strongly suggest the writing to be tightened (and more transparent), so the paper is more internally consistent and easier to follow across its four very different technical threads (soil chemistry, isotopes, organic geochemistry, microbiology).
General / major comments
1. There is no control / comparison site in this study, whose central claim is that tephra deposition causes the observed changes in SOM composition and microbial community, while presenting only one set of tephra-covered horizons; there is no non-tephra covered profile from the same area included. This makes it impossible to truly say what the effect of the tephra is (what these horizons would look like without tephra). A stronger design would have paired this site with a nearby profile that wasn't buried by ash. I do understand that's at this point impossible to correct, but it needs to be addressed.
2. Similarly to issue 1, the manuscript leans on geothermal heat from the fumarole to explain the abundance of thermophiles. However, since sample only comes from the site close to fumarole, there is no way to know whether the microbiome reflects the fumarole's influence- you would have needed to sample close to multiple fumaroles and also non-fumarole locations to claim that.
3. "Critical" microbial role but no functional data: the manuscript claims to show a critical role of microbial dynamics in soil fertility and resilience, but only reports relative abundances of bacteria and archaea (16S); there is no data on microbial activity (for example mRNA), biomass or absolute abundance, and to my understanding, relative abundances alone cannot support claims about roles in fertility / resilience.
4. Overstatement of novelty: manuscript claims to provide "key insights into mechanisms ... critical role... in soil resilience and ecosystem recovery" implying it's a new territory, when it's not (Zeglin et al. cited in this very paper, Fujimura et al. 2016, and Maltz et al. 2024 all describe comparable microbial/SOM dynamics after volcanic disturbance.). I'd suggest to reframe: novelty is in the combination of methods (Py-GC/MS + isotopes + sequencing). You might want to write a short paragraph in the introduction that would state what's already known from these papers, rather than treating the general dynamic as not established knowledge.5. Unclear replication, possibly problematic statistics: As far as I can tell, 6 samples were collected, split into 2 subsamples and then into (I think) 2 technical replicates each – meaning the "n" behind the ANOVA/Tukey letters in Table 1 is really analytical/technical replication, not biological or spatial replication. This needs to be clarified, because if I'm reading this right, running ANOVA on technical duplicates of one field sample per group is not statistically sound, and would inflate the appearance of significance. Another confusing factor is that Figure 2 reports n=3, which doesn't match the "duplicate (n=2)" described in the methods (line 122 and 130 – are these biological or technical duplicates, and are they even referring to the same duplication?). If this inconsistency is not resolved, it will read as trying to cover up a fairly weak sampling strategy.
6. Handling the contradictions around MS: results show internal mismatches specifically for the moss substrate (MS) sample: bulk %C, bulk δ13C, and the Py-GC/MS molecular profile don't agree with each other (e.g. line 191 vs. Horizon O's root density and δ13C; line 257's admission that the isotope signature doesn't match the detected compounds). Rather than treating this disagreement as worth investigating in its own right (e.g., "different methods may be sampling different pools of organic matter in this young, heterogeneous substrate"), each mismatch got its own separate, unverified fix (root exudates or microbial activity for the isotope signal; undetected LMW polysaccharides for the isotope/molecular mismatch). I would suggest the authors address this explicitly as a limitation rather than patching every separate occasion.
7. Structure and readability: as a reader, I find the Results+Discussion combined structure to make this a hard paper to follow – I for example especially struggled with the molecular composition and lipid analysis parts, as I have very little expertise in it. I think the paper would read much better structured as Results first, then a Discussion that synthesizes across layers (i.e., characterize each horizon/sample as a whole – its C/N, its metabolites, its microbiome – together), rather than the current organization. When you have such a multidisciplinary approach, it's really necessary, otherwise I imagine you to lose readers interest. I also noticed that some microbial taxa get a paragraph of commentary that doesn't really go anywhere (see Arthronema/Deinococcus comments below) – it reads as discussion for its own sake rather than because it supports an argument.
Line-by-line comments
19 / 71 – 18S rRNA gene sequencing is mentioned here and in the intro, but the methods and results only ever show 16S data. Please remove the 18S references, or add the missing data.
34 (Graphical abstract) – This is purely methodological – I can't see the actual story of the paper here, only the methods used (and even the C/N isotope analysis isn't represented). The 5 tubes for "molecular techniques" are confusing – what do they represent? 5 analyzed layers? But there were 6 sample types. Shouldn't there also be 5 (or 6) tubes for DNA sequencing, since 5 (or 6) layers were sequenced too? The bar plot for taxonomical composition is missing its 3 most abundant taxa in the legend and is hard to read as is (very small), same for the MS-related graphs – not sure what they're meant to convey as currently presented.
44 – very fine ash is technically defined as <0.063 mm, not <2 mm as stated.
52–60 – the described effects of tephra on microbial communities, plants, and SOM are quite vague, and no clear direction/trend is given; as a reader I can't tell whether fresh tephra is meant to increase or decrease nutrient retention, for example.
61 – This sentence (on PAH/condensed compound formation from ashfall–SOM interactions) feels out of place – it doesn't connect to anything before or after it in the paragraph. I'd suggest stating explicitly why PAH formation matters here, e.g. "PAHs, which are generally recalcitrant and can act as long-term soil contaminants…" and then explain why that's relevant to this study.
73 – see general comment #4 above.
84 – "Volcanism displaced to a N-S elongated…" – this sentence is missing its verb.
78 / 93 – the Las Manchas location is mentioned twice (opening sentence + again here); I'd cut the earlier one. Also, the geomorphological history of the island is quite long for a methods section – I'd move that to the introduction and keep the methods focused on the sampling site/design.
105 – this sentence feels random/out of place in its paragraph.
122 – "processed in duplicate" – biological or technical duplicates? Please clarify.
130 – same question for "duplicate analyses were performed for samples and standards" – is this referring to the same duplication as L122, or something different?
148 – were there 5 or 6 sample types? The figures throughout show 6.
157 – see general comment #5 (statistics/replication) above – this section needs a clearer description of the actual replicate structure, and the n=2 vs n=3 (Fig. 2) discrepancy needs to be explained.
163 (Table 1 caption) – a bit clumsily written, and also mixes up "p < 0.05" with "p = 0.05" as if they were two separate things, when really there's just one significance threshold. Suggested rewording: "Different letters denote significant differences between samples (one-way ANOVA with Tukey's HSD post-hoc test, α = 0.05)."
172 – "the biofilm was found in a healthy state" – what does a "healthy" biofilm actually mean here? This reads as vague. The following sentence (biofilms with C/N close to 9 having higher carbohydrate/protein content) also seems out of place – why does that matter for this study, and why is the Quorum Sensing gene mention relevant if QS wasn't actually measured here?
176 – "These findings align with…" – this sentence doesn't add anything and could be cut.
177 – this sentence is redundant given the table sits right above it and is already described in the caption.
180 – "This is the first indication of soil regeneration after the volcanic eruption of Tajogaite" is an ambitious and fairly vague claim. What's actually shown here is consistent with the well-established pattern of mosses/photosynthetic organisms as pioneer colonizers – it's a confirmation of a known pattern, not a new finding.
186 – since this is a multidisciplinary paper, I think it would help to explain in the introduction (not just assume background knowledge) how δ13C relates to carbon origin/processing. Not every reader will be familiar with this, and without it, the isotope discussion is hard to follow.
191 – the high δ13C enrichment in MS is attributed to root exudates – but Horizon O, which has the highest root density in the whole profile, shows the most negative δ13C. These two explanations can't both be true as currently framed. Also, "microbial activity linked to diverse families of microorganisms" needs to specify which microorganisms and which pathway – as it's currently written, it's an unsupported placeholder next to a citation list.
194 – the logic comparing δ13C between Horizons A/Bw and MS based on shared "tephra/lapilli" content doesn't hold up, since the samples were decarbonized before analysis – lapilli are mineral, not organic carbon. Can you clarify what pathway would let tephra/lapilli mineral content shape an organic δ13C signature?
198 – the depth-related δ13C/%C trend is described as "a key finding," but this is a well-established, previously described pattern (which the text itself acknowledges a few sentences later) – I'd suggest not opening the paragraph by calling it a "key finding."
250 – "being the second the dominant group" – needs rewording, unclear as written.
(I had a harder time following the biochemical/Py-GC/MS sections in detail – see general comment #7 on this.)
375 – this needs rephrasing: complete detection is indicated by a small gap between Chao1 and the observed richness, not by a high Chao1 value on its own (which is what's currently implied).
388 – Nitrososphaeria has been reclassified into phylum Thermoproteota since 2021 – please update.
391 – Nitrososphaeria is a class, not a genus. Also, it seems like an odd taxon to focus on for its N-cycle role, given that the data show 15N enrichment is strongest in the deeper layers, not where Nitrososphaeria is most abundant.
Figure 6A – the dominant dark blue phylum isn't identified in the legend – I assume this is Pseudomonadota given the text, but please add it, since it's the most abundant group shown.
404 – "This genus includes only two…" doesn't seem to add anything substantive – reads like filler to make the paragraph look more thoroughly discussed.
405 – it would help to show the relative abundance of Methyloligellaceae – I can't verify from the bar plots that it "dominates" horizons A, MS, and biofilm without manually looking up each genus's lineage.
L447 – cyanobacteria are described here as nitrogen fixers, which would be expected to contribute low δ15N (fixation draws on atmospheric N2) – but this doesn't match the data: MS and biofilm both show enriched δ15N, not depleted. This isn't really supported by "isotope ratios that predicted the presence of photosynthetic microorganisms" as stated.
452 – the Arthronema discussion (antitumor/antioxidant properties, phycobiliproteins) is quite speculative. The argument seems to be: other studies found Arthronema producing phycobiliproteins elsewhere - Py-GC/MS detected generic peptide markers in most samples here - therefore "the presence of phycobiliproteins cannot be ruled out." This is an overreach – generic peptide markers say nothing about a specific protein. I'd suggest cutting this passage, including the antitumor/antioxidant properties mention, which isn't relevant to the topic.
462 – the thermophile abundance is attributed to proximity to fumaroles, but again (see general comment #2), there's no comparison site without a fumarole to support this – as it stands this is speculative.
Citation: https://doi.org/10.5194/egusphere-2025-2086-RC2 -
AC2: 'Reply on RC2', Nicasio T Jiménez-Morillo, 08 Sep 2026
Opening note
Overall, I think the synthesized dataset (combining Py-GC/MS, isotopes, and 16S) is a genuinely interesting and fairly rich resource, and I find the core observation that tephra deposition alters SOM composition and microbial activity plausible. However, I am concerned about how far the interpretation is pushed beyond what a single-site, low-replication, comparison-free design can support. Furthermore, I strongly suggest the writing to be tightened (and more transparent), so the paper is more internally consistent and easier to follow across its four very different technical threads (soil chemistry, isotopes, organic geochemistry, microbiology).
Response: We sincerely thank you for your valuable comments and for your careful review of our manuscript.
General / major comments
- There is no control/comparison site in this study, whose central claim is that tephra deposition causes the observed changes in SOM composition and microbial community, while presenting only one set of tephra-covered horizons; there is no non-tephra covered profile from the same area included. This makes it impossible to truly say what the effect of the tephra is (what these horizons would look like without tephra). A stronger design would have paired this site with a nearby profile that wasn't buried by ash. I do understand that's at this point impossible to correct, but it needs to be addressed.
Response: We thank the reviewer for this important comment. We acknowledge that the absence of a nearby unaffected soil profile represents a limitation of the study and precludes a direct comparison between tephra-covered and non-tephra-covered soils. The present study was designed to characterize the biogeochemical and microbial patterns within a soil profile buried by the 2021 Tajogaite tephra, considering the different compartments of the tephra–soil system, rather than to perform a paired comparison between affected and unaffected soils. Accordingly, our interpretation is based on the differences observed among the tephra-associated materials and the underlying O, A, and Bw soil horizons. We recognize that these patterns cannot be attributed exclusively to tephra deposition, as pre-existing vertical soil heterogeneity and local geothermal conditions may also contribute to the observed variability. Nevertheless, the integrated analysis of SOM molecular composition and microbial communities across the profile provides valuable insights into the biogeochemical and microbial patterns associated with this recently tephra-covered soil system.
- Similarly to issue 1, the manuscript leans on geothermal heat from the fumarole to explain the abundance of thermophiles. However, since sample only comes from the site close to fumarole, there is no way to know whether the microbiome reflects the fumarole's influence- you would have needed to sample close to multiple fumaroles and also non-fumarole locations to claim that.
Response: We thank the reviewer for this valuable observation. We agree that, since sampling was conducted at a single site located near a fumarole, our study design does not allow us to establish a direct causal relationship between geothermal activity and the occurrence of thermophilic taxa. The proximity to the fumarole was considered as relevant environmental context when interpreting the microbial community; however, we acknowledge that the presence of thermophilic taxa cannot be unequivocally attributed to geothermal influence. As the reviewer correctly points out, a comparative sampling design including multiple fumarole-associated and non-fumarole sites would be necessary to specifically evaluate this relationship. We therefore consider our findings as a characterization of the microbial community occurring in this particular tephra-covered site under local geothermal influence.
- "Critical" microbial role but no functional data: the manuscript claims to show a critical role of microbial dynamics in soil fertility and resilience, but only reports relative abundances of bacteria and archaea (16S); there is no data on microbial activity (for example mRNA), biomass or absolute abundance, and to my understanding, relative abundances alone cannot support claims about roles in fertility / resilience.
Response: We sincerely thank the reviewer for this valuable comment and for highlighting the need to clarify the scope of our microbial data. We agree that 16S rRNA gene amplicon sequencing provides information on microbial community composition and relative abundance, but does not directly measure microbial activity, biomass, or functional processes. Accordingly, the observed shifts in microbial community composition should be interpreted as indicative of potential relationships with soil biogeochemical characteristics and development, rather than as direct evidence of microbial contributions to soil fertility or resilience. As the reviewer appropriately points out, additional approaches, such as metagenomics, metatranscriptomics, and direct measurements of microbial biomass or activity, would be required to establish these functional relationships. Nevertheless, we believe that integrating microbial community composition with SOM molecular characterization and soil physicochemical properties provides valuable insights into the associations between microbial community patterns and biogeochemical processes within this recently tephra-covered soil system.
- Overstatement of novelty: manuscript claims to provide "key insights into mechanisms ... critical role... in soil resilience and ecosystem recovery" implying it's a new territory, when it's not (Zeglin et al. cited in this very paper, Fujimura et al. 2016, and Maltz et al. 2024 all describe comparable microbial/SOM dynamics after volcanic disturbance.). I'd suggest to reframe: novelty is in the combination of methods (Py-GC/MS + isotopes + sequencing). You might want to write a short paragraph in the introduction that would state what's already known from these papers, rather than treating the general dynamic as not established knowledge.
Response: We sincerely thank the reviewer for this thoughtful comment. We agree that microbial community shifts and changes in soil organic matter dynamics following volcanic disturbance have been previously documented, as reported in the studies highlighted by the reviewer and in other works already cited in the manuscript. Therefore, we do not intend to suggest that these general post-volcanic dynamics are themselves novel. Rather, the specific contribution of the present study lies in the integrated application of complementary analytical approaches, combining Py-GC/MS, stable isotope analysis, and 16S rRNA gene sequencing to jointly characterize SOM molecular composition and microbial community structure within the recently tephra-covered soil profile of the 2021 Tajogaite eruption. Previous knowledge regarding the effects of volcanic disturbance on SOM dynamics and microbial communities is already acknowledged in the Introduction through references to previous studies on these processes. We therefore consider that the novelty of our study lies specifically in this integrated methodological approach and its application to the recently formed Tajogaite tephra–soil system. We appreciate the reviewer’s comment, as it helps to clarify the scope of the novelty claimed in our study.
- Unclear replication, possibly problematic statistics: As far as I can tell, 6 samples were collected, split into 2 subsamples and then into (I think) 2 technical replicates each – meaning the "n" behind the ANOVA/Tukey letters in Table 1 is really analytical/technical replication, not biological or spatial replication. This needs to be clarified, because if I'm reading this right, running ANOVA on technical duplicates of one field sample per group is not statistically sound, and would inflate the appearance of significance. Another confusing factor is that Figure 2 reports n=3, which doesn't match the "duplicate (n=2)" described in the methods (line 122 and 130 – are these biological or technical duplicates, and are they even referring to the same duplication?). If this inconsistency is not resolved, it will read as trying to cover up a fairly weak sampling strategy.
Response: Thanks for pointing out the mistake in Figure 2; where it says n=3, should say n=2.
- Handling the contradictions around MS: results show internal mismatches specifically for the moss substrate (MS) sample: bulk %C, bulk δ13C, and the Py-GC/MS molecular profile don't agree with each other (e.g. line 191 vs. Horizon O's root density and δ13C; line 257's admission that the isotope signature doesn't match the detected compounds). Rather than treating this disagreement as worth investigating in its own right (e.g., "different methods may be sampling different pools of organic matter in this young, heterogeneous substrate"), each mismatch got its own separate, unverified fix (root exudates or microbial activity for the isotope signal; undetected LMW polysaccharides for the isotope/molecular mismatch). I would suggest the authors address this explicitly as a limitation rather than patching every separate occasion.
Response: We sincerely thank the reviewer for point out that a reader could understand that there is a mismatch between the technics for MS sample. However, both bulk δ13C and the Py-GC/MS molecular profile agree with the presence of roots exudates: “The observed 13C-enrichment of the MS (about 4–5 ‰ above Moss, Biofilm and Horizon O samples), may be attributed to i) the heavier isotope signature of root exudates compared to other organic compounds commonly found in soil samples (Girona-García et al., 2018),” (lines 221-223) and “The presence of Campesterol and γ-Sitosterol may indicate an input of organic matter from the moss via rhizoid exudates” (lines 286-287). We will modify the text accordingly to avoid misunderstandings.
- Structure and readability: as a reader, I find the Results+Discussion combined structure to make this a hard paper to follow – I for example especially struggled with the molecular composition and lipid analysis parts, as I have very little expertise in it. I think the paper would read much better structured as Results first, then a Discussion that synthesizes across layers (i.e., characterize each horizon/sample as a whole – its C/N, its metabolites, its microbiome – together), rather than the current organization. When you have such a multidisciplinary approach, it's really necessary, otherwise I imagine you to lose readers interest. I also noticed that some microbial taxa get a paragraph of commentary that doesn't really go anywhere (see Arthronema/Deinococcus comments below) – it reads as discussion for its own sake rather than because it supports an argument.
Response: We sincerely thank the reviewer for this thoughtful and constructive suggestion. We appreciate the concern regarding readability, particularly given the multidisciplinary nature of the study and the integration of physicochemical, molecular, and microbial data. We agree that separating the Results and Discussion could provide an alternative way of presenting the information. However, we have chosen to retain the combined Results and Discussion structure because it allows each dataset to be interpreted immediately within its specific analytical and biogeochemical context, while progressively building towards an integrated understanding of the tephra–soil system. Importantly, the final integrative section brings together the physicochemical properties, SOM molecular composition, and microbial community data, providing the cross-disciplinary synthesis highlighted by the reviewer. Regarding the discussion of individual microbial taxa, these observations were included to provide ecological context for the community patterns identified across the different horizons and to support their interpretation within this recently tephra-covered soil system. We therefore consider that the current organization provides an appropriate balance between the interpretation of the individual analytical datasets and their subsequent integration. Nevertheless, we appreciate the reviewer’s suggestion and recognize that the alternative structure proposed would also represent a valid approach to presenting these multidisciplinary data.
Line-by-line comments
19 / 71 – 18S rRNA gene sequencing is mentioned here and in the intro, but the methods and results only ever show 16S data. Please remove the 18S references, or add the missing data.
Response: We thank the reviewer for pointing this out. All references to 18S rRNA gene sequencing have been removed from the manuscript.
34 (Graphical abstract) – This is purely methodological – I can't see the actual story of the paper here, only the methods used (and even the C/N isotope analysis isn't represented). The 5 tubes for "molecular techniques" are confusing – what do they represent? 5 analyzed layers? But there were 6 sample types. Shouldn't there also be 5 (or 6) tubes for DNA sequencing, since 5 (or 6) layers were sequenced too? The bar plot for taxonomical composition is missing its 3 most abundant taxa in the legend and is hard to read as is (very small), same for the MS-related graphs – not sure what they're meant to convey as currently presented.
Response: We thank the reviewer for the comments about the graphical abstract. This will be revised accordingly, following your comments and suggestions.
44 – very fine ash is technically defined as <0.063 mm, not <2 mm as stated.
Response: We thank the reviewer for pointing this out. The value for “very fine ash” has been corrected from <2 mm to <0.063 mm.
52–60 – the described effects of tephra on microbial communities, plants, and SOM are quite vague, and no clear direction/trend is given; as a reader I can't tell whether fresh tephra is meant to increase or decrease nutrient retention, for example.
Response: We sincerely thank the reviewer for this thoughtful and valuable observation. We fully appreciate the reviewer’s point regarding the complexity of the effects of tephra deposition on microbial communities, vegetation, and SOM. These responses do not necessarily follow a single directional trend, but may vary depending on tephra properties, environmental conditions, soil characteristics, and time since deposition. For this reason, the Introduction was intended to provide a general overview of the range of responses associated with tephra deposition, rather than to imply a consistent increase or decrease in processes such as nutrient retention. We appreciate the reviewer’s comment, as it highlights the complexity of these interactions and the importance of interpreting post-volcanic soil responses within their specific environmental context.
61 – This sentence (on PAH/condensed compound formation from ashfall–SOM interactions) feels out of place – it doesn't connect to anything before or after it in the paragraph. I'd suggest stating explicitly why PAH formation matters here, e.g. "PAHs, which are generally recalcitrant and can act as long-term soil contaminants…" and then explain why that's relevant to this study.
Response: Thank you for this helpful comment. We agree that the relevance of PAH formation was not sufficiently explained in the original version. We have therefore revised this sentence to better connect PAH formation with the broader context of the paragraph and to clarify its importance for this study, emphasizing that PAHs are generally recalcitrant compounds that may persist in soils and act as long-term contaminants.
73 – see general comment #4 above.
Response: Thank you for this helpful comment.
84 – "Volcanism displaced to a N-S elongated…" – this sentence is missing its verb.
Response: We thank the reviewer for pointing this out. The sentence has been revised to correct the grammatical omission and improve clarity.
78 / 93 – the Las Manchas location is mentioned twice (opening sentence + again here); I'd cut the earlier one. Also, the geomorphological history of the island is quite long for a methods section – I'd move that to the introduction and keep the methods focused on the sampling site/design.
Response: We thank the reviewer for this helpful suggestion. The redundant reference to Las Manchas has been removed, and the geomorphological description has been streamlined to avoid unnecessary detail and maintain the focus of the Methods section on the sampling site and study design.
105 – this sentence feels random/out of place in its paragraph.
Response: We thank the reviewer for this observation. This sentence was added in response to a comment from a previous reviewer to provide further clarification on this aspect. We have therefore retained it in the manuscript.
122 – "processed in duplicate" – biological or technical duplicates? Please clarify.
Response: We thank the reviewer for this question. The samples were first treated with HCl to remove any potential inorganic carbonates. After decarbonation and sample preparation, each treated sample was analysed in duplicate to determine its elemental and stable isotopic composition using EA/TCD and EA/IRMS, respectively. Therefore, the replicates reported here correspond to duplicate analytical measurements of the same processed sample rather than independent sample replicates. This procedure follows the methodology described in Jiménez-Morillo et al. (2020b).
130 – same question for "duplicate analyses were performed for samples and standards" – is this referring to the same duplication as L122, or something different?
Response: Thank you for this observation. Yes, this refers to the same analytical procedure described above, and the same approach was applied to both the samples and the standards. To clarify this point, the text will be revised as follows: “Two subsamples of each sample and standard were analysed by EA/IRMS, and each subsample was analysed in duplicate as technical replicates”.
148 – were there 5 or 6 sample types? The figures throughout show 6.
Response: We sincerely thank the reviewer for pointing out this ambiguity. To clarify, six sample types were analyzed in total: moss, the substrate beneath the moss (MS), biofilm, and three soil horizons (O, A, and Bw). We apologize for any confusion caused by the previous description.
157 – see general comment #5 (statistics/replication) above – this section needs a clearer description of the actual replicate structure, and the n=2 vs n=3 (Fig. 2) discrepancy needs to be explained.
Response: Thank you for highlighting this point. As clarified in our response to General Comment #5, the n = 3 indicated in Fig. 2 was an error and has now been corrected to n = 2. We have also revised the corresponding section of the Methods to describe the replicate structure more clearly. Briefly, each sample was divided into two subsamples, which were subjected to the corresponding sample preparation procedure. Each processed subsample was subsequently analysed in duplicate as technical/analytical replicates. The manuscript and the Fig. 2 caption have been revised accordingly to clearly distinguish between subsamples and analytical replicates and to avoid any ambiguity regarding the reported n values.
163 (Table 1 caption) – a bit clumsily written, and also mixes up "p < 0.05" with "p = 0.05" as if they were two separate things, when really there's just one significance threshold. Suggested rewording: "Different letters denote significant differences between samples (one-way ANOVA with Tukey's HSD post-hoc test, α = 0.05)."
Response: We sincerely thank the reviewer for detecting this error. The caption of Table 1 has been modified as suggested
172 – "the biofilm was found in a healthy state" – what does a "healthy" biofilm actually mean here? This reads as vague. The following sentence (biofilms with C/N close to 9 having higher carbohydrate/protein content) also seems out of place – why does that matter for this study, and why is the Quorum Sensing gene mention relevant if QS wasn't actually measured here?
Response: We thank the reviewer for this observation. The “following sentence” was meant to be an explanation of the concept “healthy” stated in the preceding sentence. To clarify, both sentences will be replaced by the following:
“The Biofilm also shows notable TOC, and its ratio TOC/N close to 9 (8.83 on average) indicates that the biofilm was found in a healthy state, since previous studies have shown that biofilms with C/N ratio close to 9 have higher concentrations of carbohydrates and proteins in the biofilm and a significant upregulation of the Quorum Sensing genes (Ramos et al., 2023).”
176 – "These findings align with…" – this sentence doesn't add anything and could be cut.
Response: We thank the reviewer for this observation. This sentence was added to clarify preceeding sentence “In contrast, the C and N values in the horizons A (17.6 ± 3.39 and 1.6 ± 0.51, respectively) and Bw (8.65 ± 4.48 and 0.95 ± 175 0.87, respectively) were consistent with those reported in different soil types, the concentration of both elements in horizon A is three-fold of those found in B (Girona-García et al., 2018; Jiménez-Morillo et al., 2020a).”, which demonstrates the correlation of our findings with previous studies. Both sentences have been modified for clarity as follows:
“In contrast, the TOC and N values in the Hhorizons A (17.6 ± 3.39 and 1.6 ± 0.51, respectively) and Bw (8.65 ± 4.48 and 0.95 ± 0.87, respectively) were consistent with those reported in different soil types. The concentration of both elements in horizon A is three-fold of those found in B, indicating a decline in TOC and N content with increasing soil depth (Girona-García et al., 2018; Jiménez-Morillo et al., 2020a).”
177 – this sentence is redundant given the table sits right above it and is already described in the caption.
Response: We thank the reviewer for this observation. This sentence was added in response to a request for clarification from another reviewer. We have therefore retained it in the text to ensure that this information is explicitly stated.
180 – "This is the first indication of soil regeneration after the volcanic eruption of Tajogaite" is an ambitious and fairly vague claim. What's actually shown here is consistent with the well-established pattern of mosses/photosynthetic organisms as pioneer colonizers – it's a confirmation of a known pattern, not a new finding.
Response: We sincerely thank the reviewer for this valuable observation. We fully agree that the role of mosses and other photosynthetic organisms as pioneer colonizers in volcanic environments is well established, as acknowledged in the manuscript through the cited literature. Our statement was not intended to present this ecological pattern as a novel finding, but rather to indicate that, within the specific context of the recently formed Tajogaite system, the observed moss colonization provides an early indication of soil regeneration following the 2021 eruption.186 – since this is a multidisciplinary paper, I think it would help to explain in the introduction (not just assume background knowledge) how δ13C relates to carbon origin/processing. Not every reader will be familiar with this, and without it, the isotope discussion is hard to follow.
180 – "This is the first indication of soil regeneration after the volcanic eruption of Tajogaite" is an ambitious and fairly vague claim. What's actually shown here is consistent with the well-established pattern of mosses/photosynthetic organisms as pioneer colonizers – it's a confirmation of a known pattern, not a new finding.
Response: We sincerely thank the reviewer for this thoughtful suggestion. We appreciate that the interpretation of δ¹³C may not be familiar to all readers, particularly given the multidisciplinary nature of the study. However, the relevance of the δ¹³C values is addressed within the corresponding Results and Discussion section, where they are interpreted in relation to carbon sources and organic matter dynamics. We therefore considered that an additional explanation in the Introduction would be somewhat redundant and have retained the current structure.
191 – the high δ13C enrichment in MS is attributed to root exudates – but Horizon O, which has the highest root density in the whole profile, shows the most negative δ13C. These two explanations can't both be true as currently framed. Also, "microbial activity linked to diverse families of microorganisms" needs to specify which microorganisms and which pathway – as it's currently written, it's an unsupported placeholder next to a citation list.
Response: Thank you for this important observation. This issue has already been raised in the comments from other reviewers and has been addressed accordingly in the revised manuscript. Specifically, the interpretation linking the relatively enriched δ13C values in the MS horizon to root exudates has been revised to avoid an apparent contradiction with the more negative δ13C values observed in Horizon O, despite its higher root density. In addition, the statement referring broadly to “microbial activity linked to diverse families of microorganisms” has been revised to avoid an unsupported generalization and to provide a more cautious interpretation consistent with the available evidence. These changes have already been incorporated into the revised version of the manuscript.
194 – the logic comparing δ13C between Horizons A/Bw and MS based on shared "tephra/lapilli" content doesn't hold up, since the samples were decarbonized before analysis – lapilli are mineral, not organic carbon. Can you clarify what pathway would let tephra/lapilli mineral content shape an organic δ13C signature?
Response: We sincerely thank the reviewer for this valuable observation. The lapilli from Tajogaite and from any previous eruption in La Palma have no carbon content. Lapilli is primarily composed of silicate minerals (like plagioclase, pyroxene, and olivine), carbon does not form part of its crystalline structure. Therefore decarbonised samples did not alter the original inorganic carbon of the lapilli. It just removed any possible carbonates that could have formed after volcanic deposition via trapped volcanic CO2 gases or carbon sequestration from atmospheric CO2. The CO2 can bind with the Ca and Mg of the lapilli minerals to form carbonates. But we are not interested in describing this inorganic process. The lapilli content of the paleo soils is mentioned because it is indicative of the volcanic origin, not because it was chemically involved in the microbial pathway of the carbon cycle. However, the similar δ13C between Horizons A/Bw and MS suggests that the recurrent lapilli deposition, and consequently burring of soil, after each volcanic eruption may have affected the carbon cycle. To better explain this in the manuscript, the paragraph have been modified as follows:
“This could be indicative of the volcanic origins of the paleo soil (Horizons A and B) (Carracedo et al., 2022) and suggests that the recurrent tephra deposition, and consequently burying of soil, after each volcanic eruption may have affected the carbon cycle during the soil formation (Matus et al., 2014 and references therein).”
198 – the depth-related δ13C/%C trend is described as "a key finding," but this is a well-established, previously described pattern (which the text itself acknowledges a few sentences later) – I'd suggest not opening the paragraph by calling it a "key finding."
Response: We sincerely thank the reviewer for this valuable observation. The sentence has been modified as suggested by the reviewer.
250 – "being the second the dominant group" – needs rewording, unclear as written.
(I had a harder time following the biochemical/Py-GC/MS sections in detail – see general comment #7 on this.)
Response: We sincerely thank the reviewer for pointing out this unclear sentence. It has been modified accordingly changinging the word “second” by “latter”, as follows:
“Only a few organic compounds were identified in the MS samples, mainly high molecular weight alkyl lipids (n-alkanes) and sterols, being the latter the dominant group (65%).
375 – this needs rephrasing: complete detection is indicated by a small gap between Chao1 and the observed richness, not by a high Chao1 value on its own (which is what's currently implied).
Response: We sincerely thank the reviewer for pointing out this important distinction. The text has been revised to clarify that the high coverage is supported by the close agreement between Chao1 estimates and observed richness, rather than by high Chao1 values alone.
388 – Nitrososphaeria has been reclassified into phylum Thermoproteota since 2021 – please update.
Response: We sincerely thank the reviewer for this important taxonomic observation. We acknowledge that the placement of Nitrososphaeria differs among current archaeal taxonomic frameworks, with some genome-based classifications placing this class within the phylum Thermoproteota. However, Nitrososphaerota was validly published as a phylum under the International Code of Nomenclature of Prokaryotes (ICNP), with Nitrososphaera as the type genus (Oren and Garrity, 2021). Within the same nomenclatural framework, Thermoproteota is recognized as a distinct phylum, with Thermoproteus as its type genus. As the taxonomic assignments in our study were performed using the SILVA 138.1 reference database, we have retained Nitrososphaerota to maintain consistency with the taxonomic framework used for sequence classification. We have included Oren and Garrity (2021) to clarify the nomenclature adopted in this study.
391 – Nitrososphaeria is a class, not a genus. Also, it seems like an odd taxon to focus on for its N-cycle role, given that the data show 15N enrichment is strongest in the deeper layers, not where Nitrososphaeria is most abundant.
Response: We sincerely thank the reviewer for this valuable observation. Nitrososphaeria has now been correctly referred to as a class rather than a genus throughout the manuscript. Regarding its role in the nitrogen cycle, our intention was not to establish a direct relationship between the relative abundance of Nitrososphaeria and the observed δ¹⁵N enrichment across the soil profile. Rather, this taxon was highlighted because members of Nitrososphaeria are well-known ammonia-oxidizing archaea and therefore potentially relevant to nitrogen cycling. We agree that the strongest δ¹⁵N enrichment occurs in the deeper horizons, where Nitrososphaeria is not most abundant. However, the discussion of this class refers specifically to its potential ecological role in the Moss, MS, and Biofilm samples, where it was detected, rather than to an explanation of the δ¹⁵N pattern across the entire soil profile. We appreciate the reviewer’s comment, as it allows us to clarify the intended interpretation of this observation.
Figure 6A – the dominant dark blue phylum isn't identified in the legend – I assume this is Pseudomonadota given the text, but please add it, since it's the most abundant group shown.
Response: We sincerely thank the reviewer for pointing out this omission. Pseudomonadota, corresponding to the dominant group shown in Figure 6A, has now been included in the legend to ensure that all major phyla are clearly identified.
404 – "This genus includes only two…" doesn't seem to add anything substantive – reads like filler to make the paragraph look more thoroughly discussed.
Response: We sincerely thank the reviewer for this observation. We consider this information relevant because Caldovatus is a taxonomically restricted genus, currently comprising only two described species, C. sediminis and C. aquaticus (Habib et al., 2017; Hu et al., 2022), both isolated from hot-spring environments and characterized as moderately thermophilic. Thus, the limited number of described species provides relevant taxonomic and ecological context for the detection of Caldovatus in the MS sample. We have therefore retained this information in the discussion.
405 – it would help to show the relative abundance of Methyloligellaceae – I can't verify from the bar plots that it "dominates" horizons A, MS, and biofilm without manually looking up each genus's lineage.
Response: We sincerely thank the reviewer for this helpful observation. We agree that the predominance of Methyloligellaceae cannot be directly assessed from Figure 6 without considering its taxonomic lineage. The figure was intended to provide an overview of the major microbial groups rather than to display every relevant family individually, and adding additional taxonomic levels would substantially increase its complexity. The statement regarding Methyloligellaceae is based on the taxonomic assignments obtained from the microbial community analysis. We have therefore retained the current representation of Figure 6 to maintain its clarity and readability.
L447 – cyanobacteria are described here as nitrogen fixers, which would be expected to contribute low δ15N (fixation draws on atmospheric N2) – but this doesn't match the data: MS and biofilm both show enriched δ15N, not depleted. This isn't really supported by "isotope ratios that predicted the presence of photosynthetic microorganisms" as stated.
Response: We sincerely thank the reviewer for this important observation. We agree that the enriched δ¹⁵N values observed in the MS and Biofilm samples should not be interpreted as evidence of active biological N₂ fixation. Our reference to Cyanobacteriota as nitrogen fixers was intended to provide ecological context based on the known metabolic capabilities of some members of this phylum, rather than to infer N₂ fixation directly from the isotopic data. We also agree that the original statement suggesting that the isotope ratios predicted the presence of photosynthetic microorganisms was too strong. The text has therefore been revised to clearly separate the taxonomic evidence for the presence of Cyanobacteriota from the interpretation of the isotopic data. The δ¹⁵N values are now discussed more cautiously as reflecting the integrated effects of different nitrogen sources and transformation processes, rather than as a direct indicator of N₂ fixation. Likewise, the occurrence of photosynthetic microorganisms is supported by the microbial community data rather than inferred from the isotope ratios alone.
452 – the Arthronema discussion (antitumor/antioxidant properties, phycobiliproteins) is quite speculative. The argument seems to be: other studies found Arthronema producing phycobiliproteins elsewhere - Py-GC/MS detected generic peptide markers in most samples here - therefore "the presence of phycobiliproteins cannot be ruled out." This is an overreach – generic peptide markers say nothing about a specific protein. I'd suggest cutting this passage, including the antitumor/antioxidant properties mention, which isn't relevant to the topic.
Response: We sincerely thank the reviewer for this thoughtful observation. We fully agree that the peptide markers detected by Py-GC/MS cannot be attributed specifically to phycobiliproteins, and we did not intend to suggest that the presence of these compounds was demonstrated in our samples. Rather, this discussion was included to provide additional ecological and biochemical context for Arthronema, a cyanobacterial genus detected in the studied samples and previously reported to produce phycobiliproteins and other bioactive compounds. The statement that “the presence of phycobiliproteins cannot be ruled out” was deliberately framed as a possibility rather than as evidence of their occurrence, since Py-GC/MS does not allow identification of specific proteins. Similarly, the reference to the reported antitumor and antioxidant properties of Arthronema was included as complementary information on the known biochemical potential of this genus, rather than as a functional inference from our data. We therefore consider that retaining this information provides broader biological context for the occurrence of Arthronema while explicitly acknowledging the analytical limitations of the Py-GC/MS data.
462 – the thermophile abundance is attributed to proximity to fumaroles, but again (see general comment #2), there's no comparison site without a fumarole to support this – as it stands this is speculative.
Response: We thank the reviewer for this observation. As acknowledged in our response to General Comment #2, the present sampling design does not allow a causal relationship between fumarolic activity and thermophile abundance to be established. The proximity to the fumarole is therefore considered as relevant environmental context for interpreting the occurrence of thermophilic taxa at this particular site, rather than as definitive evidence of geothermal control. We appreciate the reviewer’s comment and acknowledge that this interpretation should be considered within the limitations of the sampling design.
Citation: https://doi.org/10.5194/egusphere-2025-2086-AC2
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AC2: 'Reply on RC2', Nicasio T Jiménez-Morillo, 08 Sep 2026
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