the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 08 Oct 2024
Bacteria as paleoenvironmental proxies: the study of a cave Pleistocene profile
Abstract. Caves are well-known archives that preserve valuable information about the past, relevant for reconstructing past climates and environments. We sampled sediments from a 480 cm deep profile. 16S rRNA gene-based metabarcoding analyses were undertaken that complemented lithological logging, sedimentology, and Optically Stimulated Luminescence (OSL) dating. These analyses revealed different sedimentation conditions along the profile with various water inputs. The OSL age of the sediments places the profile between 74.7 ± 12.3 to 56 ± 8 ka (base to top). However, more recent reworking processes (during LGM – Last Glacial Maximum paleofloods) in the upper and lower passages of the cave might have occurred. Bacterial compositions changed with depth, from soil bacteria (present in the upper part of the sediment profile) to thermophilic/sulfurous bacteria (abundant in the deeper samples of the profile). Considering the thermophilic bacteria, we could only assume their origin from a surface of hot sulfurous, old thermal springs, or sapropel sediments.
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RC1: 'Comment on egusphere-2024-2973', Anonymous Referee #1, 23 Oct 2024
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Haidău and co-authors in their study demonstrate the potential of bacteria as paleoenvironmental proxies in caves, especially in caves where traditional biological indicators are absent. The shift in bacterial composition with depth, from soil-specific to thermophilic and sulfur-associated bacteria, suggests changes in deposition conditions and water sources through time. This research highlights the need to consider bacteria as a valuable tool in multi-proxy paleoenvironmental reconstructions.
During the review I encountered a few issues with the MS. Firstly, by reading the MS I was not convinced that the authors fully consider how contamination and natural processes that happen after burial (i.e. taphonomic processes) might have affected the bacterial DNA and might have provide them with mixed results, although the expertise and experience of the authors is undoubted, I would be more convinced if parts of the text are rewritten highlighting the above. The MS will benefit if authors try to explore how this "sediment reworking" could impact the bacteria found at different depths.
I also believe that it should be stated in the manuscript how they tried to prevent contamination when collecting samples. This methodology/measures of protection can be valuable for others trying to perform such sampling. Also, the absence of this information might raise doubts about the accuracy of the findings, since modern bacteria could easily contaminate older sediment layers.
Another issue, in my opinion, is that the authors mainly base their idea of a hot spring as the source of thermophilic bacteria on indirect evidence. They haven't convincingly ruled out other possibilities, for example bacteria traveling long distances through epikarst.
The authors don't fully discuss the limitations of using bacteria to reconstruct ancient environments. While the study shows great potential, it's important to acknowledge the challenges of interpreting bacterial data. For example, bacteria can go dormant, and their DNA can persist long after they're dead. This could mislead researchers about what the environment was like.
The manuscript also doesn't consider how other environmental factors, like pH levels, nutrients, and even other microbes, might have shaped the bacterial communities. The manuscript would be more informative if it explored the functions of the bacterial communities. The authors briefly mention the roles of some bacteria in natural cycles, but they don't thoroughly examine the metabolic capabilities of the communities and how these might reflect past conditions. The manuscript's analysis could be improved with more advanced statistical methods to confirm the differences between bacterial communities found at different depths. Finally, although I do understand that sampling microbes in a cave sequence is extremely demanding, I think that 5 samples only for a sequence 480 cm long covering ~20 k yrs are not enough to extract secure findings. I would appreciate it if the authors added to the text a small explanatory part explaining why they selected only 5 samples.
I believe that if all the above issues highlighted here addressed adequately by the authors the MS should be considered for publication from the journal.
Citation: https://doi.org/10.5194/egusphere-2024-2973-RC1 -
CC1: 'Reply on RC1', Oana Moldovan, 04 Nov 2024
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Publisher’s note: the content of this comment was removed on 5 November 2024 since the comment was posted by mistake.
Citation: https://doi.org/10.5194/egusphere-2024-2973-CC1 -
CC2: 'Reply on CC1', Oana Moldovan, 04 Nov 2024
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We hope our contribution to Biogeosciences will be published in this revised form. The revised manuscript marks all the changes in red.
Based on your suggestions, the following are some comments concerning the changes made to the manuscript.
Haidău and co-authors in their study demonstrate the potential of bacteria as paleoenvironmental proxies in caves, especially in caves where traditional biological indicators are absent. The shift in bacterial composition with depth, from soil-specific to thermophilic and sulfur-associated bacteria, suggests changes in deposition conditions and water sources through time. This research highlights the need to consider bacteria as a valuable tool in multi-proxy paleoenvironmental reconstructions.
During the review I encountered a few issues with the MS. Firstly, by reading the MS I was not convinced that the authors fully consider how contamination and natural processes that happen after burial (i.e. taphonomic processes) might have affected the bacterial DNA and might have provide them with mixed results, although the expertise and experience of the authors is undoubted, I would be more convinced if parts of the text are rewritten highlighting the above.
The PMP2 profile and that part of the cave do not contain bones or taphonomic context. In the Material section, we mentioned the bones, and in Mirea et al. (2021), such taphonomic studies are described. Still, all the work was done in PMP1 (see Fig. 1d), representing a completely different situation than the studied PMP2. A few details were added in the text that will hopefully better describe the position of PMP2.
The MS will benefit if authors try to explore how this "sediment reworking" could impact the bacteria found at different depths.
The sediment reworking processes in Muierilor Cave are examined by Mirea et al. (2021), especially for PMP1, where the chronology established secondary depositions. In PMP2, there is no evidence for reworking or re-deposition; it is only for the input of sediments during key events like the paleofloods that occurred during warmer and wetter conditions in the region. No other significant processes, such as bioturbation or collapses, were identified. We agree that the term was not used properly and was deleted or replaced where PMP2 sediments were discussed.
I also believe that it should be stated in the manuscript how they tried to prevent contamination when collecting samples. This methodology/measures of protection can be valuable for others trying to perform such sampling. Also, the absence of this information might raise doubts about the accuracy of the findings, since modern bacteria could easily contaminate older sediment layers.
We added a paragraph to explain how contamination was avoided.
Another issue, in my opinion, is that the authors mainly base their idea of a hot spring as the source of thermophilic bacteria on indirect evidence. They haven't convincingly ruled out other possibilities, for example bacteria traveling long distances through epikarst.
The Polovragi-Cernadia karst area (where Muierilor Cave is carved) spans approximately ~2.5 km², with the limestone bar's width ranging from 2 km to 0.8 km and its thickness between 150 m and 250 m. Given this isolation of the limestone within surrounding magmatic and metamorphic rocks, it is unlikely that bacteria were transported over long distances (no more than 2 km) via the epikarst network. Moreover, PMP2 is in an area of the cave with no proof of present or former percolation and no speleothems. It is near a former siphon between two different cave passages. We added a phrase in the Material section to make this clear.
The authors don't fully discuss the limitations of using bacteria to reconstruct ancient environments. While the study shows great potential, it's important to acknowledge the challenges of interpreting bacterial data. For example, bacteria can go dormant, and their DNA can persist long after they're dead. This could mislead researchers about what the environment was like. The manuscript also doesn't consider how other environmental factors, like pH levels, nutrients, and even other microbes, might have shaped the bacterial communities. The manuscript would be more informative if it explored the functions of the bacterial communities. The authors briefly mention the roles of some bacteria in natural cycles, but they don't thoroughly examine the metabolic capabilities of the communities and how these might reflect past conditions. The manuscript's analysis could be improved with more advanced statistical methods to confirm the differences between bacterial communities found at different depths.
The extraction and high-throughput methods we used can identify active and dormant taxa. Testing the metabolism of bacteria can be misleading on such old sediments and it was not the idea of this study. The idea of the project was to investigate the bacterial diversity of a dated profile and compare the data with other proxies. If the results had been confusing and comparing different proxies impossible, more questions could have been proposed. Nevertheless, we consider that the results were informative for some of the events in the past, and a new figure (Fig. 4) with a statistical clustering was added. We also agree that future studies can consider more analyses/ experiments such as the ones recommended and make our study more interesting.
Finally, although I do understand that sampling microbes in a cave sequence is extremely demanding, I think that 5 samples only for a sequence 480 cm long covering ~20 k yrs are not enough to extract secure findings. I would appreciate it if the authors added to the text a small explanatory part explaining why they selected only 5 samples.
We sampled every 50cm in depth, but not enough genetic material could be extracted for the samples at -100 and -250 cm, most likely due to the high amount of clay, or other inhibitors that we could not remove. Details are given in the text, and we often face the problem of inhibitors in old and new cave sediments.
I believe that if all the above issues highlighted here addressed adequately by the authors the MS should be considered for publication from the journal.
Thank you for the comments and suggestions that improved the manuscript.
In the name of all authors, Dr. Oana Moldovan
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CC2: 'Reply on CC1', Oana Moldovan, 04 Nov 2024
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CC1: 'Reply on RC1', Oana Moldovan, 04 Nov 2024
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