A Timeline of Bacterial and Archaeal Diversification in the Ocean

Reviewer #1 (Public Review):

Martinez-Gutierrez and colleagues presented a timeline of important bacteria and archaea groups in the ocean and based on this they correlated the emergence of these microbes with GOE and NOE, the two most important geological events leading to the oxygen accumulation of the Earth. The whole study builds on molecular clock analysis, but unfortunately, the clock analysis contains important errors in the calibration information the study used, and is also oversimplified, leaving many alternative parameters that are known to affect the posterior age estimates untested. Therefore, the main conclusion that the oxygen availability and redox state of the ocean is the main driver of marine microbial diversification is not convincing.

Basically, what the molecular clock does is to propagate the temporal information of the nodes with time calibrations to the remaining nodes of the phylogenetic tree. So, the first and the most important step is to set the time constraints appropriately. But four of the six calibrations used in this study are debatable and even wrong.

(1) The record for biogenic methane at 3460 Ma is not reliable. The authors cited Ueno et al. 2006, but that study was based on carbon isotope, which is insufficient to demonstrate biogenicity, as mentioned by Alleon and Summons 2019.

(2) Three calibrations at Aerobic Nitrososphaerales, Aerobic Marinimicrobia, and Nitrite oxidizing bacteria have the same problem - they are all assumed to have evolved after the GOE where the Earth started to accumulate oxygen in the atmosphere, so they were all capped at 2320 Ma. This is an important mistake and will significantly affect the age estimates because maximum constraint was used (maximum constraint has a much greater effect on age estimates and minimum constraint), and this was used in three nodes involving both Bacteria and Archaea. The main problem is that the authors ignored the numerous evidence showing that oxygen can be produced far before GOE by degradation of abiotically-produced abundant H2O2 by catalases equipped in many anaerobes, also produced by oxygenic cyanobacteria evolved at least 500 Ma earlier than the onset of GOE (2500 Ma), and even accumulated locally (oxygen oasis). It is well possible that aerobic microbes could have evolved in the Archaean.

Once the phylogenetic tree is appropriately calibrated with fossils and other time constraints, the next important step is to test different clock models and other factors that are known to significantly affect the posterior age estimates. For example, different genes vary in evolutionary history and evolutionary rate, which often give very different age estimates. So it is very important to demonstrate that these concerns are taken into account. These are done in many careful molecular dating studies but missing in this study.

Reviewer #2 (Public Review):

In this paper, Martinez-Gutierrez and colleagues present a dated, multidomain (= Archaea+Bacteria) phylogenetic tree, and use their analyses to directly compare the ages of various marine prokaryotic groups. They also perform ancestral gene content reconstruction using stochastic mapping to determine when particular types of genes evolved in marine groups.

Overall, there are not very many papers that attempt to infer a dated tree of all prokaryotes, and this is a distinctive and up-to-date new contribution to that oeuvre. There are several particularly novel and interesting aspects - for example, using the GOE as a (soft) maximum age for certain groups of strictly aerobic Bacteria, and using gene content enrichment to try to understand why and how particular marine groups radiated.

Comments:

One overall feature of the results is that marine groups tend to be quite young, and there don't seem to be any modern marine groups that were in the ocean prior to the GOE. It might be interesting to study the evolution of the marine phenotype itself over time; presumably some of the earlier branches were marine? What was the criterion for picking out the major groups being discussed in the paper? My (limited) understanding is that the earliest prokaryotes, potentially including LUCA, LBCA and LACA, was likely marine, in the sense that there would not yet have been any land above sea level at such times. This might merit discussion in the paper. Might there have been earlier exclusively marine groups that went extinct at some point?

What do the stochastic mapping analyses indicate about the respective ancestors of Gracilicutes and Terrabacteria? At least in the latter case, the original hypothesis for the group was that they possessed adaptations to life on land - which seems connected/relevant to the idea of radiating into the sea discussed here - so it might be interesting to discuss what your analyses say about that idea.

I very much appreciate that finding time calibrations for microbes is challenging, but I nonetheless have a couple of comments or concerns about the calibrations used here:

The minimum age for LBCA and LACA (Nodes 1 and 2 in Fig. 1) was calibrated with the earliest evidence of biogenic methane ~3.4Ga. In the case of LACA, I suppose this reflects the view that LACA was a methanogen, which is certainly plausible although perhaps not established with certainty. However, I'm less clear about the logic of calibrating the minimum age of Bacteria using this evidence, as I am not aware that there is much evidence that LBCA was a methanogen. Perhaps the line of reasoning here could be stated more explicitly. An alternative, slightly younger minimum age for Bacteria could perhaps be obtained from isotope data ~3.2Ga consistent with Cyanobacteria (e.g., see https://pubmed.ncbi.nlm.nih.gov/30127539/).

I am also unclear about the rationale for setting the minimum age of the photosynthetic Cyanobacteria crown to the time of the GOE. Presumably, oxygen-generating photosynthesis evolved on the stem of (photosynthetic) Cyanobacteria, and it therefore seems possible that the GOE might have been initiated by these stem Cyanobacteria, with the crown radiating later? My confusion here might be a comprehension error on my part - it is possible that in fact one node "deeper" than the crown was being calibrated here, which was not entirely clear to me from Figure 1. Perhaps mapping the node numbers directly to the node, rather than a connected branch, would help? (I am assuming, based on nodes 1 and 2, that the labels are being placed on the branch directly antecedent to the node of interest)?

Author Response:

We thank the editors and reviewers for their time in reviewing our manuscript. We would like to post a brief response to the peer reviews at this stage, and we will revise the manuscript and re-post at a later time.

The main concerns regarding our molecular dating approach consist of the limited number of marker genes used for phylogenetic reconstruction, the molecular clock model employed, and the calibrations used. Firstly, regarding the marker genes that we used in our phylogenetic reconstruction, we will point out that we have extensively benchmarked these methods in a previous study (Martinez-Gutierrez and Aylward, 2021). We initially planned on presenting all of these results together in the same manuscript, but we decided that benchmarking phylogenetic marker genes across all Bacteria and Archaea together with an extensive molecular dating analysis was too much for a single study, and we therefore divided the results into two papers. In short, we agree with R1 that the use of different marker genes will lead to marked differences in the posterior ages of our Bayesian molecular dating analysis; however, we demonstrated that several of the few marker genes shared between Bacteria and Archaea lack of a strong phylogenetic signal and therefore introduce topological biases in the final phylogeny (i.e., long branch attraction). Consequently, using poorly-performing marker genes for molecular dating does not add valuable information to the overall analysis.

Secondly, regarding the autocorrelated Log-normal model used in our study (-ln on Phylobayes), we believe this is appropriate. Besides being biologically meaningful for our study, it represents a compromise between a relaxed model with rate variation across branches and the assumption of correlation between parent and descent branches (Thorne et al., 1998). In contrast, a fully uncorrelated model that assumes rate independence across branches would make our analysis extremely time-consuming and intractable given our study encompasses all of Bacteria and Archaea. Nonetheless we understand the concerns raised, and in a future manuscript we will include age estimates resulting from the CIR and UGAM models in order to explore the potential effect of model selection in posterior dates.

Thirdly and lastly, we will point out that calibrations for molecular dating of Bacteria and Archaea are always highly controversial, and there are essentially no calibrations for the early evolution of life on Earth that would not be contested to some degree. Researchers are therefore left to use their best judgment and provide reasonable rationale, which we have done here. We understand that strong opinions abound in this area, and many researchers will disagree with our approach, but that alone does not invalidate our study. Moreover, the main novelty of our approach is the use of a large tree that combines Bacteria and Archaea; extensive benchmarking of different calibration points on such a large tree is not possible here as it may be on a smaller set. One of the main concerns is the use of the age estimate of the Great Oxidation Event (GOE, 2.4 Ga) as minimum and maximum constraints for oxygenic Cyanobacteria, and Ammonia Oxidizing Archaea and aerobic Marinimicrobia, respectively. We agree that oxygen may have existed before the GOE as proposed previously (e.g., Ostrander et al., 2021), however; the strongest geochemical evidence so far (Mass Independent Fractionation of Sulfur, MIFs, (Farquhar et al., 2000)) indicates a significant accumulation of oxygen around that time. We therefore feel that this is a reasonable calibration to use for microbial lineages that have a physiology that is tightly linked to the production or consumption of oxygen. Similar reasoning has been used in other molecular dating studies, so our logic is not out of step with much research in the field (Liao et al., 2022; Ren et al., 2019).

Due to the limitations of molecular dating studies of microorganisms, we have been very careful to avoid strong conclusions based on the absolute dates we calculated, and the primary interest of readers will likely be the relative divergence times of the marine clades we study (i.e., the overall timeline of microbial diversification in the ocean). We will provide a more in-depth assessment of models and calibrations for Bacteria and Archaea in a future draft, but in the meantime we hope to convey that our study is not without merit despite the substantial challenges of research in this area.

References:

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• Liao T, Wang S, Stüeken EE, Luo H. 2022. Phylogenomic Evidence for the Origin of Obligate Anaerobic Anammox Bacteria Around the Great Oxidation Event. Mol Biol Evol 39. doi:10.1093/molbev/msac170
• Martinez-Gutierrez CA, Aylward FO. 2021. Phylogenetic Signal, Congruence, and Uncertainty across Bacteria and Archaea. Mol Biol Evol 38:5514–5527.
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