Review of Brittingham et al.

“Locally Produced Sedimentary Biomarkers in High-Altitude Catchments Outweigh Upstream River Transport in Sedimentary Archives”

by Joseph Novak (jobnovak@ucsc.edu)

Summary and overall significance

Brittingham et al. present a study of higher plant n-alkane transport in a small stream in the Armenian Caucasus. Their analysis of n-alkane distributions and isotopic composition in stream sediments and soils revealed that, contradictory to expectation, stream sediments did not have an n-alkane composition that approximated the area-weighted upstream vegetation. This is a valuable contribution to the field that provides needed insight into how the transport of n-alkanes may imprint upon sedimentary archives utilized for paleoclimate or palaeoecological study. Overall, the manuscript’s main findings are well-supported by the data, and I look forward to the article’s publication after revision to address my comments and technical corrections.

Recommendation: minor revision

General comments:

My main comment on the manuscript is a request that the authors include information about their mixing model, including necessary equations and values used for constants, as a subsection in the methods. The values for constants could be added as a table or in the text. The additional text and equations would substantially clarify the discussion in section 4.2. and, more importantly, are necessary for evaluating the comparison between measured and expected values shown in Figure 6. Much of the necessary text can be moved from lines 194–208 to the methods section.

I also strongly recommend appending the supporting data for this study as supplementary tables or as files uploaded to a publicly accessible database. The data may prove useful for future paleoclimate or palaeoecological work in this region, so it is important to make sure the data is accessible to those who may need it in the future.

Lastly, I recommend inserting more references to the figures within the text. As is, each figure is only referenced once or twice, even in sections where the data is discussed in detail. More figure references will make the text easier to follow.

Specific comments

Title

I suggest amending the title to better reflect the findings and conclusions of the study. As is, the title appears to suggest that the findings here are applicable to all high-altitude river (and lake?) catchments. However, this assertion is not made in the manuscript.

Two other things to point: (1) n-alkanes measured in this study should really be referred to as leaf wax or vegetation biomarkers since they are produced by plants, not produced by sediments; (2) I believe you mean downstream river transport, as in away from the river’s headwaters. As written, the word upstream implies transport of sediment in the opposite direction of the water flow, which I do not think is what you mean. 

A potential alternative title could be:

“Locally Produced Leaf Wax Biomarkers in the High-Altitude Lesser Caucuses Outweigh Downstream Transport”

Abstract

Lines 18–22: these first two sentences are a bit repetitive. I suggest removing the first sentence that reads, “Sedimentary records…signals.” and starting the abstract with the second sentence that reads “The integration of…”. This still introduces the study effectively while making the language more succinct.

Line 25: I suggest removing “the existence of” and just stating “We utilize a treeline…”

Lines 35–36: Please clarify the language here, I am unsure what you mean by the “latter observation” since the language in lines 32–35 makes it seem like you do not have sufficient evidence to distinguish which mechanism is driving the pattern you observe in the stream sediments (this is also what I took away from reading the rest of the manuscript).

Line 38: I am unclear what “changes in upstream fractionation differences” means here. I suspect you are talking about fractionation of leaf wax hydrogen isotope composition caused by changes in landscape vegetation, please clarify the language as this confused me a bit.

Introduction

Line 45: Is “so called” necessary here?

Line 49: I suggest adding a sentence here briefly summarizing any paleoclimate records from this region that utilize leaf wax n-alkanes if there are any as that will clarify the importance of this study to your readers.

Line 62: I suggest replacing “though” with “although”

Line 64: I suggest rewriting as “The carbon isotope (δ¹³C) composition of plant tissue is primarily set by the photosynthetic pathway of the plant.

Line 72: Could you please comment on which of these factors is important for your study site? For example, are there C₄ plants in the Caucuses?

Lines 83–86: I suggest rewriting this sentence to summarize the common findings of the studies you cite here. For example, I recall that the Feakins et al. 2018a study cited here found that riverine n-alkanes approximated area-weighted vegetation in the Amazon River catchment (the opposite of your finding, which highlights what makes your study interesting). Perhaps these other studies have found the same thing, or mixed results?

Lines 99–102: I suggest rewriting as “Comparison of the hillside and streambed sedimentary n-alkanes allows…”. The current wording is a bit repetitive and clunky.

Lines 104–108: I suggest splitting into two sentences, revising to: “…Tornero et al., 2016). Pleistocene sediments…”

Lines 118–119: I suggest removing the phrase “In order to extract n-alkanes” as it is repeating the end of the previous sentence.

Lines 120–121: please describe the solvent schedule, water content of the silica gel, and quantity of silica gel used for chromatographic separation.

Line 123: please describe any internal and external standards used for sample quantification. Please clarify whether and how the different response factors of the C₂₅ – C₃₃n-alkanes were accounted for during sample quantification or in calculation of the OEP and ACL values. This is important because, while the peak area of each compound is proportional to concentration, that relationship (response factor) is mass-dependent and so is slightly different for each of the n-alkanes.

Line 125: Did you forget to insert a citation here?

Line 136: How exactly did you correct for size effects? Also, I think you want to use the standard error of the mean, not standard deviation. In general, Polissar and D’Andrea (2011) [https://doi.org/10.1016/j.gca.2013.12.021] is an excellent guide on how to calculate uncertainties associated with leaf wax stable isotope measurements, particularly hydrogen isotopes.

Results

Line 144: could you please provide some example chromatograms or histograms showing the n-alkane distributions in your samples? This could be as a supplementary figure if you prefer.

Lines 144–145: I suggest reporting the range of carbon preference index (CPI) values of your samples to support this statement.

Line 149: please replace “averages” with “is”

Lines 150–151: Please replace “average” with “mean”

Lines 151–153: Please specify what kind of statistical test was done here and the N samples or degrees of freedom. Same goes for the test mentioned in lines 155–156.

Line 161: please replace “average” with mean

Lines 170–171: does the word “significant” imply a statistical test was done here? If so, please specify what kind, the p-value, and N samples or degrees of freedom

Lines 173–174: I see, this is what is referred to in 170–171. Please consider combining and condensing into a single sentence to clarify the language / keep the text concise.

Line 177: Please specify the N samples or degrees of freedom

Lines 178–179: Please revise to: “δD values were also more negative in stream sediment samples collected above the treeline (-175‰) than those collected below the treeline (-158‰).”

Please also specify the statistical test, p-value, and N samples / degrees of freedom

Line 180: Please revise to “…stream sediment samples…”

Discussion

Lines 194–208: as said in my general comments, please move these lines to a new section of the methods that includes the equations and constants used in the mixing model. In addition, please specify where the satellite images used for your mapping came from with appropriate references.

Line 209: I recommend beginning your revised section 4.2. here. Also, I suggest replacing “this mixing model” with “our mixing model”

Line 211: range of expected values for which measurement(s)? Please clarify.

Line 212: Please reference a figure to back up this assertion

Lines 213–214: Please also reference a figure here. Additionally, this is not really how ACL values (I assume that is what you are talking about here, please clarify) are used in practice. They tend to be thought of as a more qualitative indicator of vegetation / ecosystem composition. I would suggest rewriting this sentence to discuss that the ­n-alkane distributions do not show an expected “mixed” signal but are rather indistinguishable from the endmember values.

Line 223: I suggest replacing “though” with “although”

Lines 236–241: Please clarify the language here. I had to reread this section several times to understand what you mean.

Lines 245–254: this is a good idea, but the implementation here could use some improvement. More information is needed about how the lines in Figure 7 were calculated, as is the “expected” δD timeseries and the relative timing of the vegetation shift. It may be useful to expand this exercise into its own section in the Discussion with an accompanying short section in the Methods, but this is really just a suggestion.

Line 251: I suggest replacing “heavily affected” with “influenced”

Lines 255–258: I think this is better suited to the Introduction. Please see my comment regarding lines 83–86.

Conclusions

No comments

References

Line 294: why is this centered and not aligned to the left? Also, should it not be bolded and called “references”?

In general, please check the formatting of your citations and ensure that all article titles have proper typesetting (subscripts, superscripts, Greek characters, etc.), that journal names are italicized, and check for typos.

Figures

In general, please ensure to upload 300 DPI or higher images or vector files of the images. The current images are a bit fuzzy.

Figure 1: the points showing your sample sites in the right panel are very difficult to see. Could they be larger or a different shape? Perhaps a shape with a black border would help.

Figure 2: in the figure caption the samples are referred to as sediments while on the figure they are referred to as soils. Please make these consistent with each other. I also recommend switching to a colorblind friendly color palette (red-green is particularly hard to distinguish for many colorblind people).

Figure 3: Please remove the lines. They really clutter up the figure and make it difficult to see the pattern that is very clearly evident in the data (really nice result!). Also, I recommend making this figure square to match the style of figure 2. Finally, please consider using three different symbol shapes and a colorblind-friendly color palette.

Figure 4: please also remove the lines here. Same comments as figure 3 regarding the color palette and symbol shapes. Also, please use δ¹³C for your y-axis label for consistency with Figure 2.

Figure 5: please also remove the lines here. Same comments as figure 3 regarding the color palette and symbol shapes. Also, please use δD for your y-axis label for consistency with Figure 2.

Figure 6: Could this figure be made a little larger? It is very hard to read in this small format. Also, the low DPI of the image is really evident here. Please be sure to replace with a higher quality image. I also recommend switching to a colorblind friendly color palette and using different symbol shapes for the different panels.

Figure 7: please see my comment regarding lines 245–254 of the text

Technical Corrections

Line 34: Typo, shrubs is misspelled as “shurbs”

Line 47: Typo here, “supports” should be “supporting”

Line 78: Missing space between period and first word of following sentence “…(Gamarra et al., 2016).The fractionation…”

Line 172: Typo, “averages” should be “average”

Line 174: lower case delta is needed, not an upper case delta. It does not matter that this is the beginning of a sentence, the upper case delta means something different than the lower case delta and is not applicable here.

Lines 177–178: Typo here, sentence is divided by a random period

This study, by Brittingham et al, investigates the influence of transport and depositional processes on sedimentary lipid biomarker records (leaf-wax n-alkanes and their Hydrogen and Carbon isotopic composition) by analyzing soil and stream sediments across a 1000 m altitude gradient spanning the closed deciduous forest, treeline ecotone and alpine meadow vegetation belts in a first-order catchment located in the Areguni Mountains, Armenia. Main results show that, while there is a major difference in the soil and stream n-alkane and their isotopic values above and below the treeline, stream sediment biomarkers below the treeline predominantly reflect local vegetation rather than upstream contributions. This finding is important for the interpretation of sediment biomarker records, in that it shows that processes at the level of the catchment must also be accounted for and may critically influence the distribution of different biomarker compounds in sediment archives.

The manuscript reads very well, aims are clear, the topic approached is relevant and addresses an important knowledge gap in the field of biomarker-based palaeoenvironmental and palaeoclimate reconstructions. Some further clarifications are needed in the overall design of the study and interpretation of the results (details below), but most of these are minor. The main recommendation for the authors is to describe the study area more thoroughly, in a separate section, and provide information on temperature and precipitation patterns, geology and soil types (for example, are soils acidic?) and dominant vegetation species for each of the vegetation belts, because in my opinion this information is relevant for interpreting biomarker distribution. Then, I think it is important to show at least some of the more representative chromatograms (these may be even placed in the supplementary material). Also, there is an issue that, in my opinion, needs to be further expanded in the discussion: if the top 10 cm of soil were removed prior to subsampling for biomarker analysis (L. 116), this means that the collected soil samples likely do not represent modern vegetation, as soil takes a long time to form. Conversely, stream bed sediments may be of a more recent age compared to the soil samples, as stream beds are highly dynamic environments. How could this potential age discrepancy impact the results? 

I therefore recommend the manuscript for publication, provided that these clarifications are addressed.

Minor comments

Title: could be adjusted a bit, because it suggests a generalized conclusion, while the results are study-case based, and it is not clear to what extent these findings can be extrapolated to all high-altitude catchments.

Introduction:

L.45-48 A word is missing from this sentence: “and Iran, ‘that’ supports a wide variety…”?

L.49-54 The link between interpretation of palaeoclimatic records in the study region and the environmental signal of biomarkers in sedimentary archives is unclear. I suggest adding a sentence to explain why understanding processes involved in the sedimentary integration of biomarkers, and the scale of biomarker environmental signals are relevant for palaeoclimatic reconstructions.

L 64-72 Why are the carbon isotope values of C3 vs C4 plants relevant for the study area or for the aims of this study? E.g., were there major shifts in the importance of C3 vs C4 plants in the vegetation history of the area? Is the proportion of the C4 plants in the current vegetation increasing?

L.73 Same as in the previous comment, it is hard to grasp the relevance of hydrogen isotope values in leaf-wax n-alkanes for the study area or for the aims of this study.

L.83-86 Could you, please, summarize some of the key findings of the referenced publications, that are also relevant for this study?

L.105 What proxies were used to assess the relationships between the past treeline and climate? Are there knowledge gaps that remained unaddressed and that are addressed within the present study?

L.108 Regarding the potential of sediments at Kalavan to reconstruct the treeline-climate relationship, I assume it refers to a biomarker-based reconstruction, because it is not clear. But then, why would biomarkers be the preferred proxy instead of more established proxies, like for example plant macro-remains and pollen? Justification needs to be a bit stronger here.

Methods:

I recommend the authors to begin with a subsection which describes the study area in terms of climate, geology, soil types and dominant vegetation species for the two main vegetation belts and the treeline ecotone. I also recommend the authors to create another section, that could be placed last, that collates the description of the statistical methods used (significance tests and mixture models).

L.119-120 Please provide a reference for the Soxhlet procedure used for lipid extraction. What intrigues me is the relatively high proportion of methanol in the solvent mixture and the long extraction time.

L .121 Please specify what solvent or solvent mixture was used for n-alkane separation.

L.121-125 Please add details on: oven temperature, use of blanks to test for lab contamination, standards used for n-alkane quantification, method used for integration of peak areas etc.

L.125 ‘REF’ shows a missing reference?

Results:

L.143-145 It would be great to see some of the most illustrative chromatograms added to the supplementary file. This would help the reader understand better the n-alkane distribution in different sets of samples.

L.174 Please, add the design of the significance test to the methods section.

L.152 word missing: ‘between the average values of the “n-alkane?” above treeline and below…’

I don’t see any description of results obtained for the mixing model.

Discussion:

L.184-186 Please, reference the relevant figures here.

L.194-208 Consider moving these paragraphs in a separate section of the methods, where you could also include information about the statistical tests used. But overall, I very much like the idea of using a mixing model to compare expected and obtained biomarker compound values.

L.200 What does the phrase ‘tree and grass sediment’ refer to?

L.210-212 Please reference the relevant figure for this statement.

L.212 I assume ‘deciduous-sourced n-alkanes’ refers to deciduous trees, but it’s a bit ambiguous, because there are also deciduous herbaceous plants. Could you also include what the dominant deciduous species in the forest are?

L.228 Please include the distance from the study site of the lacustrine core that was analyzed for pollen.

L 233-234 This general statement needs a reference.

L.261-263 As an additional research direction, perhaps collecting water samples for lipid analysis could help clarifying the role of transport and depositional processes.

Figures: most of them are blurry and should be uploaded in a better resolution.

Fig. 1 Could you, please, specify the source of the satellite images? Also, I would find it more relevant if the figure included a close-up of the studied catchment with sampling points superposed on vegetation types.

Figures 3-5. I assume the green and red rectangles are soil samples (although it is not clear, and also not colorblind-friendly). But it should also be clarified which of the stream samples (blue triangles) were taken from above and from below the treeline respectively.

Figure 6. Please make it larger, and also increase the resolution, because the labels are hardly visible.