Peer review of: Undinarchaeota illuminate DPANN phylogeny and the impact of gene transfer on archaeal (2020)

Reviewer #1 (Remarks to the Author): 

In this article, Dombrowski and colleagues describe how they obtained 6 good quality (medium to 
high) genomes from publicly available metagenomes (MAGs) for the “Uncultivated Archaeal Phylum 2” 
(UAP2) group of archaea. This group is interesting as previously described as deeply-branching within 
Archaea, in particular in the debated clan of “DPANN. The authors analysed the obtained MAGs 
together with 6 previously described MAGs of UAP2, and propose a thorough phylogenomic analysis. 
They provide a robust phylogenetic analysis based on several marker genes sets, taking into account 
several possible sources of phylogenetic incongruences and artifacts. In order to extract marker genes 
that are most suitable to build species phylogenies, they propose an original ranking system based on 
the recovery of largely accepted phylogenetic groups. The controlled phylogenetic reconstructions 
repeatedly resulted in the positioning of UAP2 as an independent lineage within a DPANN clan. They 
therefore propose to elevate to the UAP2 at the phylum level under the name “Udinarchaeota”. Their 
phylogenetic proximity and proposed monophyly with the so-called “Cluster 2” within the DPANN clan 
was proposed, and further supported by shared genomic traits. This study also offers an assessment 
of the Udinarchaeota metabolic capacities that suggest they are mostly fermentative and might 
engage in symbiosis, even though hosts could not be clearly identified. 

Overall, this study offers important insights into deep Archaeal phylogeny (clanhood of DPANN and 
positioning of UAP2), while shedding light on a little understood, newly discovered DPANN lineage that 
is very likely to mostly include symbionts. The article is well-written and clearly reflects a considerable 
amount of work. The conclusions seem supported by the data and analyses. I also would like to 
acknowledge the authors for the efforts put in making the extensive datasets and tools used, widely 
available to the community. 

Main comments 

- I believe the main text should better reflect the amount of work invested in phylogenetic analyses. It 
is mostly discussed how taking into account compositional biases showed the robust positioning of 
Udinarchaeaota as an independent lineage, close to DPANN Cluster 2. Biases in per-site substitution 
rates were also investigated by removing fast-evolving sites, but the outcome is not discussed in 
section on lines 179-191. Even though results were consistent throughout sites removal, this should 
be stated as it demonstrated further robustness in the phylogenetic positioning of Udinarchaeota (and 
maybe no impact from per-site rate variations?). 

- Regarding the phylogenetic approach taken to rank marker genes by phylogenetic incongruence, I’m 
wondering if the marker genes having the lowest rankings have mostly supported incongruences, or if 
they just rather suffer from a lack of phylogenetic signal. These would represent very different reasons 
for their “unsuitability” for inferring species phylogenies. Did the authors look into this systematically? 
I believe a brief discussion about this would be interesting for many in the field given how widely 
these gene sets are being used, on top of what is discussed in Supp Mat (lines 89-102). 

- On the same note, regarding the detection of horizontal gene transfers in section on line 330 
(“Insights into interaction partners”), I believe that it would be more convincing if some clear cases of 
horizontal gene transfers were to be shown in trees/discussed even briefly in Sup Mat. For instance, 
could the family discussed on line 356-357 be shown in a tree? 

- It would have been interesting to see in how many other lineages the 520 core genes had detected 
homologs. I guess all core genes don’t offer the same power to detect HGT given their taxonomic 
distribution in other lineages? Maybe could this be added to Table S16 as the number of lineages 
where the core gene was found? The number of Udinarchaeota genomes harbouring each core gene 
could also be specified here. By the way, I don’t understand why there are more than 520 lines on 
that table? I can see that each marker appears several times? Does this reflect the different 
observations from bootstrap trees? 

- The authors attempted to search hosts with a technique that I believe which might favour the 
detection of conserved, shared hosts across Udinarchaeota. Could this be that these species have 
developed specific interactions with different hosts along their diversification? In which case, could this 
explain why no HGT route is observable, unlike for other DPANN lineages? What is the author’s view 
on this aspect? 

- Line 365-366, it is mentioned that Udinarchaeales could co-occur with some Dehalococcoidales. 
Could the authors find any signs of potential HGT between Chloroflexi and Udinarchaeota? How likely 
do the authors think it is that the potentially exchanged genes are not part of the core genes 
analysed? 

Other comments 

- Line 149: it is said that the “best-fitting models of sequence evolution” were used, yet it does not 
seem that this was statistically assessed e.g. using the “–m TEST” option of IQ-Tree. Maybe just say 
they are among the “most sophisticated models”? 

- Line 151: I haven’t seen any mention of statistical topology testing? Where did these analyses take 
place in the study? To test which topology against which? 

- Line 476: “We mined…” specify that the authors searched for Udinarchaeota? 

- Line 494: To screen for contaminants, it is said that Diamond was used to assess taxonomic origin, 
but in Sup Mat, it is said line 34 that Blastp was used. So which is it? 

- Line 570: I don’t understand what is meant by “combining TIGRFAM and GTDB”? What's the goal 
here? How is it ensured that TIGRFAM families meet the pre-requisites to build species trees 
(universality + unicity in genomes)? I believe some details are missing here. 

- In the Methods, I believe that the numerous supplementary tables should be referred to on more 
occasions, in order to help the reader find the datasets mentioned along the text. For instance, section 
starting on line 518: where is the list of genomes used as the “backbone dataset”? Section starting on 
line 559: refer to the tables S4 and S5? etc… 

Figures 

- Figure 1: The text in the box is inconsistent with the legend as it is said in the legend: "40% of the 
most heterog. sites were removed". So which is it? 

- Figure 2: The legend could be improved by mentioning what's represented in grey. It seems it 
corresponds to what's missing from all genomes. 

- Figure 3 and Supp Figures 58-61: Indicating the Cluster 2 of DPANN on these figures could help 
(with brackets?) or instead, just mention in the figures legends that they are above Udinarchaeota 
within the DPANN rectangle?  

- Figure 4: It is confusing, as there is a title to the figure in the legend "Potential HGTs..." and one on 
the figure: "Phylogenetic relationships of Undin. core proteins". I think the title in the legend is 
misleading. It's not only showing potential HGTs, but overall phylogenetic relatedness of the genes. 
Some could be interpreted as HGTs. I'd rather use the title displayed on the figure, that is more 
reflective of the figure content.  

- Table 1. In the table legend, “parentheses” should replace “brackets”. 

Typos 

line 71: “artefacts”, line 82: “artifacts” => pick one? 
line 71: “branch” 
line 132: “marker” 
line 388: “affected” 
line 695 “sequences” missing in “with <1000” 

Reviewer #2 (Remarks to the Author): 

This manuscript makes an impressive contribution to our understanding of DPANN evolution and 
ecology and is clearly the product of extensive and well performed analysis, even if most of it ended 
up in the huge supplementary information section. Furthermore, the analysis of HGT and how it 
affects the phylogeny of currently disputed archaeal groups is highly valuable for the field. 
I am not sure why in the age of online only publications manuscripts must be kept so short, and there 
are sections in the supplementary information, especially those describing gene content, that should 
have been in the main text, IMHO. 

I have only minor points, see below. 

"Nanoarchaeota are ectosymbionts of TACK archaea" - it would be better here to mention the specific 
lineages within TACK or at least spell out TACK for the reader. 
Table 1 - strain heterogeneity is not clearly presented 
"low ranking makers were highly incongruent" - should be "... markers..." 
"clan" and "clanhood" are relatively recent terms in phylogenetics that most readers will be unfamiliar 
with and should be introduced. 

Since the archaeal root cannot be reliably inferred are there synapomorphies supporting DPANN as a 
clan? 

"Nanohaloarchaeota and Halobacteria shared a large amount of analyzed 345 proteins horizontally 
(12.4% and 7%)" - unclear what the two different % refer to in this sentence. 

Either figure 4 is not adequately explained or there are lineages for which there are more proteins that 
originate from HGT than by shared ancestry (for example the Halobacteria). Such cases have been 
shown in bacteria (for example the Thermotogales), but not in archaea, so if this is indeed the case for 
multiple lineages the text should say something about it.