Review Reports - Geomicrobial Investigations of Colored Outer Coatings from an Ethiopian Rock Art Gallery

Round 1

Reviewer 1 Report

General comments:

The manuscript “Geomicrobial investigations of colored outer coatings from an Ethiopian rock art gallery”, submitted to Coatings by Li-Wu et al., is a well done and very interesting study. This ms provides interesting information useful understand whether different rock samples from the Yabelo paintings collected in close proximity may reveal coatings with different minerology and biology. Please, see below my few specific comments.

Specific comments:

Lines 26 – 27: Arrange the key words in alphabetic order

Line 36: Delete [Error! Reference source not found.]

Lines 68 – 76: Highlight your hypothesis and predictions

Lines 79 – 92: Provide the geographical coordinates of your study areas

Lines 97 – 125: You should better explain your methods. Currently it is unclear how you have carried out your experiments. I think that you should provide more details in this section. Don’t forget that your experiments should be replicable by anyone

Line 213: The name of the phylum and genus od the bacteria strains is unreadable

Author Response

Reviewer #1

Specific comments:

Lines 26 – 27: Arrange the key words in alphabetic order

In the revised version of the paper, the keywords have been arranged in alphabetic order. Please see lines 27-28.

Line 36: Delete [Error! Reference source not found.

The phrase has been deleted. Please see line 37.

Lines 68 – 76: Highlight your hypothesis and predictions

Following the Reviewer’s suggestion, the text of the revised manuscript has been improved as follows:

Lines 66-70: “Our hypothesis is that coating types associated with the Yabelo paintings may show different chemical and biological compositions, despite being exposed to the same environment. In fact, the host rock provides the same moisture sources with the same chemical content to the rock coatings. Since the parental rock and the environmental exposure are the same, the contribution of microbial activities to the coatings’ genesis should be considered.”.

Lines 79 – 92: Provide the geographical coordinates of your study areas

The site is not yet under protection; for that reason, in agreement with local authorities, we preferred not to provide the precise geographical coordinates of the study site. In many rock art sites, including some Ethiopian contexts, unauthorized independent researchers run invasive sampling of rock art paintings. However, we added a map in figure 1 (panel a) indicating the position of the studied site.

Lines 97 – 125: You should better explain your methods. Currently it is unclear how you have carried out your experiments. I think that you should provide more details in this section. Don’t forget that your experiments should be replicable by anyone

Following the Reviewer’s comment, the text of the revised manuscript has been improved as follows:

(Lines 133-145) “Rock samples, conserved in the stabilization solution DNA/RNA Shield (Zymo Research), were processed few days after their collection. Total genomic DNA were extracted according to the manufacturer's instructions of PowerBiofilm DNA Isolation Kit (MoBio Laboratories Inc., Carlsbad, CA). High-throughput sequencing analysis of the V3–V4 region of the bacterial 16S rRNA gene (primers CS1_341F/CS2_806R) was performed by using a MiSeq platform (Illumina) with v3 chemistry providing 2 x 300 paired-end reads [24]. Raw data were pre-processed, quality filtered, trimmed, de-noised, paired, and modelled via QIIME2 [25] and DADA2. Chimeras were detected using DADA2 according to the “consensus” method [26]. Sequences were clustered into Amplicon Sequences Variants (ASV). ASVs were assigned using a Naïve-Bayes classifier trained on the SILVA database [27]. Multi packages of R software (R Core Team, Version 3.3.0, Vienna, Austria) [28] were used to calculate the alpha diversity, to generate the rarefaction curves and the Venn diagram. Samples were rarefied at the minimum library size of 92380. The rarefaction curves were drawn using the function “rarefaction” [29] from the vegan R-package [30].”.

Line 213: The name of the phylum and genus od the bacteria strains is unreadable.

In agreement with the Reviewer’s comment, figure 6 has been modified to make the phyla and genus readable.

Author Response File: Author Response.pdf

Reviewer 2 Report

The paper entitled “Geomicrobial investigations of colored outer coatings from an Ethiopian rock art gallery" is a very interesting and novel study. The authors have conducted a study to evaluate two coatings of rock art gallery using microscopic and molecular techniques. I consider this article to be very suitable for publication in coatings journal. However, here are some small suggestions to be considered by the authors.

The authors should better explain and justify why they chose the two types of coating in the study and not others. What is the real interest of these two types of caotings studied? These aspects should be explain better in abstract and/or introductions sections.
Line 36. Reference not found
Line 73. I would change “rare” for “novel”
Line 88-92. Again, explain better the selection of two types of samples.
Line 105 “100 weight % oxide”
Line 111: “/”
Line 124-125. Detail the names of R packages.
Table 1. There are 11 cases of sample 1 and 3 cases of sample 2. Why these differences and the samples are not compensated?

Author Response

Reviewer #2

The authors should better explain and justify why they chose the two types of coating in the study and not others. What is the real interest of these two types of caotings studied? These aspects should be explain better in abstract and/or introductions sections.

We collected several samples from the Yabelo site. However, the two coatings investigated were the most interesting because (i) they belonged to two panels of the rock wall bearing a huge quantity of rock art and (ii) they cover the major part of the rock wall.

The text of the revised manuscript has been improved as follows:

(Lines 94-98) “Among them, we selected two samples representative of the two most common coatings present on the rock wall at YAB6, and from the most problematic panels in terms of conservation. These panels also present a high concentration of paintings; for sake of heritage conservation, we collected samples at the margin of panels, far from paintings.”.

Line 36. Reference not found

The reference has been added. Please see line 37.

Line 73. I would change “rare” for “novel”

The change has been made. Please see line 78

Line 88-92. Again, explain better the selection of two types of samples.

The text of the revised manuscript has been improved as follows:

Line 105 “100 weight % oxide”

The correction has been made. Please see line 118.

Line 111: “/”

The correction has been made. Please see line 125.

Line 124-125. Detail the names of R packages.

Details have been added. Please see line 142.

Table 1. There are 11 cases of sample 1 and 3 cases of sample 2. Why these differences and the samples are not compensated?

SEM-EDS sampling points are distributed to identify the differences in the mineral constituents of each coating. In this case, sample 2 is very homogeneous and therefore it does not require a high number of sampling points. On the contrary, the coating of sample 1 is more complex and consisting of many different mineral constituents. For that reason, we performed more data collection on sample 1.

Author Response File: Author Response.pdf

Reviewer 3 Report

This is an iterating article coming physicochemical characterization of ancient rock painting with microbiology accompanying it. The authors describe two samples and analyze their composition and microbiome. It is interesting that bacterial communities differ. I just wonder about possible human contamination during specimen collection - can you please describe in detail the sampling procedure in the MM section. Step by step how sample was collected, what sort of instruments and equipment was used (was it sterile?). how long it was from sample collection to the analysis, what was the storage temperature (was it minted at the site temperature, snap frozen?). Please comment accordingly in the discussion. Some references are missing.

Author Response

Reviewer #3

I just wonder about possible human contamination during specimen collection - can you please describe in detail the sampling procedure in the MM section.

Samples were collected by using gloves and sterile chisels. Samples for DNA investigations were stored in sterile falcon tubes containing the stabilization solution DNA/RNA Shield (Zymo Research) to preserves the genetic integrity at ambient temperatures. The samples have been processed few days later, as soon as they arrived in Italy.

This information has been added in the text of the revised version at lines 101-104 and 133-134.

Step by step how sample was collected, what sort of instruments and equipment was used (was it sterile?). how long it was from sample collection to the analysis, what was the storage temperature (was it minted at the site temperature, snap frozen?). Please comment accordingly in the discussion.

Please see question #15.

Some references are missing.

References have been checked throughout the text of the revised version.

Author Response File: Author Response.pdf

Reviewer 4 Report

A very nice work. Perhaps conclsuion could be written as bullets, one per each highligth,

Author Response

Reviewer #4

A very nice work. Perhaps conclusion could be written as bullets, one per each highlight

According to the Reviewer’s suggestion, the conclusion has been written as bullet points. Please see lines 342-357.

Author Response File: Author Response.pdf

Reviewer 5 Report

The manuscript is written well, however, I have one major concern, few major and few minor remarks. I recommend major revision.

Major concern: The sampling of Ethiopian cultural heritage was invasive (L88: "Several samples were taken from the shelter with chisels..."). Did you receive a permission from local (or federal) authorities for sampling, exporting and analyzing the artifacts? If yes, this must be clearly stated. In L348, you acknowledged the "Authority for Research and Conservation of Cultural Heritage (Addis Ababa)", however, their role is unclear. Clarification requested.

Major remarks:

1. Materials and methods: A map with exact location of the sampling site, a brief description of methods (not only references) would be appreciated.

2. DNA isolation method missing. Please provide all necessary details in the whole materials and methods section.

3. please explain all acronyms

4. It is unclear how you received phenotype information from 16S RNA analysis (Fig 7). Based on Illumina sequencing, you were able to identify max to the genus level, and since you did not perform any metabolic tests, the listed phenotypes are very speculative. I would recommend you either to remove this figure and reformulate the discussion section, or provide supporting data.

Minor remarks:

Fig1a: scale or approximate gallery size missing.

L72 and throughout: please replace "microflora" with "microbiota

Fig 2: Please insert the space between values and units, and use the greek letter mu/my for prefix micro

L176: *fungal structures

Throughout the manuscript: please insert non-breaking space between values and units

Fig 4: see Fig 2

Fig 3: please magnify the text next to the scale bar

Rarefaction curves: method missing

Microbial communities: how many subsamples per sample did you analyze? Only one library per sample?

Author Response

Reviewer #5

Major concern: The sampling of Ethiopian cultural heritage was invasive (L88: "Several samples were taken from the shelter with chisels...").

We underline that the sampling was poorly invasive because was not performed in correspondence of paintings, but in marginal parts of panels with drawings.

Following the Reviewer’s suggestion, the revised text has been improved as follows:

(Lines 96-98) “These panels also present a high concentration of paintings; for sake of heritage conservation, we collected samples at the margin of panels, far from the paintings”.

Did you receive a permission from local (or federal) authorities for sampling, exporting and analyzing the artifacts? If yes, this must be clearly stated. In L348, you acknowledged the "Authority for Research and Conservation of Cultural Heritage (Addis Ababa)", however, their role is unclear. Clarification requested.

The work has been performed under the aegis of the Authority for Research and Conservation of Cultural Heritage (Addis Ababa). The same Institution granted all the permissions for fieldwork, sampling, export and analyses of samples. Moreover, an official of the Authority for Research and Conservation of Cultural Heritage supervised our sampling.

To explain this, the following sentences have been added to the revised manuscript:

(Lines 372-376) “Field missions to Ethiopia are part of the project “Tracing the first Herders in East Africa: Cultural, Symbolic and Biological Trajectories” (permit n.14/2006) directed by Dr. Marina Gallinaro. The Authority for Research and Conservation of Cultural Heritage (Addis Ababa) granted to MG all necessary permits for sampling, exportation, and analyses of samples described in the study, which complied with all the applicable regulations.”.

Moreover, Prof. E.E. Spinapolice has been added among the authors. She is the leader of the (H)ORIGN project and granted field operations in Ethiopia, including sampling of the YAB6 site.

Major remarks:

Materials and methods: A map with exact location of the sampling site, a brief description of methods (not only references) would be appreciated.

The method section has been improved.

DNA isolation method missing. Please provide all necessary details in the whole materials and methods section.

In agreement with the Reviewer’s comment, the text of the revised manuscript has been improved as follows:

please explain all acronyms

All the acronyms have been explained throughout the text.

It is unclear how you received phenotype information from 16S RNA analysis (Fig 7). Based on Illumina sequencing, you were able to identify max to the genus level, and since you did not perform any metabolic tests, the listed phenotypes are very speculative. I would recommend you either to remove this figure and reformulate the discussion section, or provide supporting data.

The text of the revised paper has been improved as follows:

(Lines 224-226) “After a bibliographic research, the ASVs were classified according to eight main categories reported in Figure 7. The reads allocated to each category were presented as a heatmap to show the main features of the biofilm communities in samples 1 and 2.”.

(Line 298-299) “From the taxonomic assignment, the main futures of the community can be inferred, and the associations between the mineralizing metabolisms and the rock coating mineralogy can be obtained.”.

Minor remarks:

Fig1a: scale or approximate gallery size missing.

The scale bar has been added to figure 1b.

L72 and throughout: please replace "microflora" with "microbiota

The correction has been made.

Fig 2: Please insert the space between values and units, and use the greek letter mu/my for prefix micro

Figure 2 has been improved.

L176: *fungal structures

The correction has been made.

Throughout the manuscript: please insert non-breaking space between values and units

The non-breaking space between values and units has been added throughout the text of the revised manuscript.

Fig 4: see Fig 2

Figure 4 has been improved.

Fig 3: please magnify the text next to the scale bar

SEM images are those produce by the scanning microscope and there is no way to enlarge the writings. The latter represents the setting of the instrument. For sake of clarity, large scale bars have been added.

Rarefaction curves: method missing

Following the Reviewer’s comment, the text of the revised manuscript has been improved as follows:

(Lines 144-145) “Samples were rarefied at the minimum library size of 92380. The rarefaction curves were drawn using the function “rarefaction” [29] from the vegan R-package [30].”.

Microbial communities: how many subsamples per sample did you analyze? Only one library per sample?

The manuscript reported the results obtained from one library per sample. Unfortunately, given the nature of our research with protected rock art, we were unable to analyze more than one sample. Furthermore, the sample sizes were very limited, therefore, subsamples were not possible. This is a very common situation for research in cultural heritage, where a destructive sampling is required.

Author Response File: Author Response.pdf

Round 2

Reviewer 5 Report

I have read the author's replies, and they addressed all remarks. Most importantly, they addressed my major concern. This manuscript is ready for publication.