of 23
1
Peer Review Information
Journal
:
Nature Microbiology
Manuscript Title:
Unique mobile elements and scalable gene flow at the prokaryote
-
eukaryote boundary revealed by circularized Asgard archaea genomes
Corresponding author name(s):
Fabai Wu
Reviewer Comments & Decision
s
:
Decision Letter, initial
version:
Dear Victoria, dear Fabai,
Thank you for your patience while your manuscript "Evolutionary Plasticity of Archaea at the
Eukaryotic Root" was under peer
-
review at Nature Microbiology. It has now been seen b
y 3 referees,
whose expertise and comments you will find at the end of this email. As you will see from their
comments, although they find your work of potential interest, they have raised a number of concerns
that will need to be addressed before we can c
onsider publication of the work in Nature Microbiology.
In particular, you will see that while all referees praise the enrichments, new genomes and the
analyses based on these data, they also make multiple suggestions for improvement, particularly in
term
s of streamlining the main messages and performing some additional analyses to further
substantiate some of the main claims. For example, referee #1 suggests revising the placement using
a more comprehensive set of publicly available genomes (or otherwise
tone down the current claims
accordingly); referee #2 suggests testing more formally whether the core archaeal genes are playing
essential conserved roles in Asgards, whereas gene acquisitions from bacteria and ESPs are more
niche
-
relevant or generally les
s conserved; referee #2 also says it may be useful to quantify the
support for an Asgard origin of these genes in eukaryotes using approximately
-
unbiased tests, or
another appropriate tree selection test; and referee #3 suggests additional analyses to supp
ort the
claim that the mobilome is involved in eukaryogenesis through passage of genes from bacteria to the
nascent eukaryotic cell. As you will see, referee #3 also suggests some microscopy
-
based assessment
of the enrichments, particularly in light of pas
t work documenting protrusions in Promethearchaeum,
yet we would like to clarify that although we agree that such analyses may be interesting and
informative, they would not be required for subsequent consideration of your manuscript for
publication in Nat
ure Microbiology.
In addition to these requests for additional analyses, the referees also make several suggestions on
2
how to streamline the paper
-
these seem quite clear and straight forward to address. Should further
experimental data and text modifica
tions allow you to address these criticisms, we would be very
happy to look at a revised manuscript. We are committed to providing a fair and constructive peer
-
review process, so please do not hesitate to contact us if there are specific requests from the
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*****************************************************
Reviewer Comments:
Reviewer #1 (Remarks to the Author):
Overall,
this study is a very interesting read and innovative, and the reconstruction of two complete
Heimdallarchaeota genomes is an important contribution to our understanding of the Asgard archaea.
I do believe it deserves to be in Nature Micro, however there ar
e some fairly issues that need to be
resolved:
The authors state that these complete genomes are “phylogenetically closest to eukaryotes”, however
their phylogenies only include very few of the dozens of genomes available. It has been suggested
4
that Heimd
all are closest to eukaryotes in various other publications, but this is not well resolve yet. It
is an open question, and a very important one! Their phylogeny supports this; however, it doesn’t
include a comprehensive set now publicly available, there ma
y be other lineages not included that
may be more closely associated with the eukaryote branch. I know first
-
hand this is not a small
undertaking (to resolve the position of eukaryotes) with many genomes. So, I would back off on these
statements, or run ad
ditional phylogenies with more MAGs.
I have concerns about the identity of some of the CRISPR/Cas
-
containing contigs (shown in figure 2
and in text ln 203). MAG B51_G16 (GCA_003650225.1) is a Chloroflexi bacteria and Figure 2f
B53_G16 (GCA_003660845.1) is
a Pacearchaeota. Both are from NCBI PRJNA362212 and associated
Guaymas Basin publications. Both of these identities are supported by Dombrowski et al. 2018 and
GTDB. Perhaps these contigs were miss
-
binned in the original publication, or is there co
-
infect
ion
occurring? This is something that should be investigated and explained more.
Sample location for homologs of AagV1
-
7. In Figure 2a (E44_bin34 or GCA_004376455.1) “homologs
of the AagV1
-
7 protein” and in text, pg. 9 and reference 13 is for the Eastern
Gulf of Mexico, Atlantic
Ocean (26.28 N 86.81 W)
*****************************************************
Reviewer #2 (Remarks to the Author):
In this manuscript, Wu et al. sequence, assemble and analyse 8 new Asgard archaea genomes, and
perform comparativ
e analyses that bear on archaeal genome evolution and eukaryogenesis.
The most significant contributions of the manuscript, all of which are in my view of major importance,
are:
(i) the generation, through culture enrichment, of two new complete Heimdalla
rchaeota genomes, but
the six other high
-
quality new genomes are also very welcome and useful data for understanding
Asgard biology and studying questions around eukaryogenesis;
(ii) comparative genomic analyses of these high
-
quality genomes that confirm p
revious findings that
Asgards encode various "eukaryote
-
specific" (or eukaryote
-
related) proteins, and may have donated
these to eukaryotes via the Asgard
-
derived host cell for the mitochondrial endosymbiont (Note:
though in some respects confirmatory, thi
s kind of confirmation is actually very important for the field
at the moment, given ongoing debates about the quality of the published Asgard MAGs).
(iii) The identification and bioinformatic analysis of virus
-
like integrated elements with intriguing
biol
ogy in Heimdallarchaeota, and more generally an analysis of the "mobilome" of these Asgards,
which is now possible thanks to the high quality of these assemblies relative to published MAGs.
This is an excellent and timely analysis that will make a real di
fference in ongoing discussions about
eukaryogenesis, but which also provides interesting new data on the genome biology of Asgards. In
general, the methods are appropriate (caveat: those that I am qualified to judge, particularly the
phylogenetics and com
parative genomics) and the interpretations are well
-
founded. Some more
detailed/specific comments follow.
1. Lines 289
-
90: The eukaryote root and the placement of the eukaryote nuclear lineage within the
Asgard archaea are distinct concepts (with the euka
ryote root usually used to refer to ideas about the
deepest split within eukaryotes, not their relation to archaea or bacteria). I suggest reformulating here
5
to avoid confusion, e.g. "This is in agreement with recent analyses placing the eukaryotic nuclear
lineage/host cell for the mitochondrial endosymbiont within the Asgard archaea" or "This is in
agreement with recent analyses supporting a close specific relationship between eukaryotes and
Heimdallarchaeota", or similar.For the same reason, it may be wor
th re
-
considering the title of the
manuscript to make clear that it is eukaryogenesis/the relationship of eukaryotes and Archaea, and
not the inter
-
relationships among eukaryotic groups, that is being addressed
2. Lines 315
-
322: The parallel drawn between
the finding of ESPs and especially bacteria
-
origin genes
acting as a kind of accessory "cloud" of genes and the situation in eukaryotes is interesting, but needs
to be unpacked and set out with greater specificity and detail. In eukaryotes, the finding th
at species
with fewer genes tend to be enriched for archaeal
-
origin genes has been explained either by the
increased functional importance of archaeal
-
origin genes (e.g. as informational genes), or alternatively
due to loss of the mitochondrion (and so a l
arge tranche of bacterial
-
origin genes) in the most reduced
eukaryotic lineages such as Microsporidia. Presumably, the hypothetical mechanistic basis here is the
former: that the core archaeal genes are playing essential conserved roles in Asgards, whereas
gene
acquisitions from bacteria and ESPs are more niche
-
relevant or generally less conserved. If so, this
should be stated clearly and, if possible, tested against the data presented in Figure 4. Do the
functions of the 900 core archaeal genes differ from
those of the bacteria
-
origin genes? How does the
situation compare to the "half
-
lives" of horizontally
-
acquired genes more generally, which are often
lost soon after they are acquired (e.g. van Passel et al. 2008
https://journals.plos.org/ploscompbiol/art
icle?id=10.1371/journal.pcbi.1000059)
3. In Figure 4(c), I found the schematic of the new hypothesis unclear. The "ancestral Asgard"
presumably existed before the first Heimdall
archaea, yet the ancestor is shown as donating genes
along the FECA to LECA stem. Perhaps this would be clearer if the donor were labelled as "related
Asgard archaea" or similar (if this is the intended meaning...).
4. One of the exciting proposals in th
e study is that some of the bacterial
-
origin genes in eukaryotes
might have been acquired via the archaeal host, or one of its relatives (i.e., an "indirect" acquisition
from bacteria). However, the gene trees provided in Extended Figure 10 in support of t
hese individual
cases are, with the exception of (d), perhaps not very compelling. This is not surprising given the
difficulty of inferring robust single gene trees, particularly for anciently diverged sequences, but these
results should perhaps be discuss
ed more cautiously in the main text. It might be useful to quantify
the support for an Asgard origin of these genes in eukaryotes using approximately
-
unbiased tests, or
another appropriate tree selection test. The likelihood of the ML tree in which the Asg
ard+eukaryote
sequences (or a subset of them) form a clade to the exclusion of Bacteria could be compared to the
ML tree in which the closest bacterial group forms a clade with the eukaryotes.
Minor points:
1. Define "mobilome" in the abstract when first
used, or reformulate to avoid this perhaps field
-
specific term.
2. In Extended Figure 8, I think the author of the 2013 study was "Alvarez
-
Ponce".
*****************************************************
Reviewer #3 (Remarks to the Author):
6
The paper by W
u and colleagues reports further interesting genomic insights into the Asgard archaea.
They enriched two members of Heimdallarchaeota, currently the clade that is closest to eukaryotes
and named them Ca. Heimdallaerchaeum endolithica and Ca Heimdallarchae
um aukensis. This allowed
them to obtain complete circularized genomes from these two strains.
This is a very important result as isolation of Asgard representatives is one of the highest priorities in
the archaeal field. Presently, as far as I understand
, only one enriched and sequenced member of
Asgard in available (Promethearchaeum).
The paper is therefore surely an important one, but my general impression is that it lacks focus, and
does not really know which way to go. Even the abstract does not real
ly reflect the content, as a large
part of the results is focused on proviruses and mobile elements. I commend the authors for wanting
to make a new scenario for the emergence of eukaryotes, but I fear that this does not rely strongly on
the analyses and i
s not discussed in detail. In this respect, the very title of the paper does not reflect
the content of the article.
The main message to me is that the authors obtained two enrichments and they analyzed in detail the
mobilome, and this is original enough t
hat the paper could be clearly built around it. The rest of
results are presented as confirmations of previous data (placement with respect to, eukaryotes,
presence of ESPs). Moreover, if the authors want to claim that this mobilome is involved in
eukaryog
enesis through passage of genes from bacteria to the nascent eukaryotic cell, this must be
supported much more by the data and discussed in depth, clearly showing that these mobilomes do
vehicle genes from bacteria to eukaryotes (e.g., that some of these g
enes are embedded in mobile
elements? by one clear phylogeny as main figure?). This could be an extremely powerful message.
Also, I was disappointed that the authors have two beautiful enrichments in their hands but do not
attempt at more detailed microsco
py data. This could be extremely interesting to see for example, if
the same protrusions are observed as in Promethe.
I might be wrong, but I think the authors are not citing nor comparing their results to those of the
recent Nature paper by Koonin and Men
g Li. This is important.
Finally, I found the figures complex (figure 2 in particular) and not very well explained in the text.
Below a few comments to help improve readability and impact.
This is a personal impression, but I feel that the text is struct
ured somehow erratically.
I think the first thing the readers want to see is a tree where these two genomes are placed, and this
instead comes very late in Figure 3.
Then I would move on to describe what is new in terms of ESPs in these genomes (basically
move
here the paragraph from lines 265
-
303), and then move on to the most original results (the mobilome
and the gene flux), which would nicely lead to the proposed hypothesis (although substantiated by
more analysis).
Also, the first paragraph is very h
ard to follow in the absence of a main figure.
Line 102, this could be a new paragraph presenting a description of the genome contents. Here, there
is a sentence that could be completed by some discussion: line 116, what is the missing enzyme for
ester
-
li
nked lipids for? what does its absence imply? It is also absent from other Asgard genomes? The
authors mention a mosaic distribution of these enzymes but do not really discuss it.
7
Line 132: why is this intriguing?
Line 137: did you try to reassemble by u
sing your method?
Line 143: say more clearly that they are from bacteria and mention which bacteria.
A large part of the results presents an extensive analysis of integrated proviruses in the two circular
genomes. This is very interesting and as I said,
should be highlighted more in both the title and the
abstract. Lines 250
-
256, please include a main figure and more detailed results to support your claims
at lines 256
-
258).
Line 305, the analysis of gene flux scaling is nice and new, and could certainly
be further developed.
The possibility that the genome sized of the other MAGs are affected by assembly problems is not
evoked.
Line 330: interesting model, which could be merged with the last paragraph as a common discussion.
However, as I said earlier,
I do not think it is strongly supported by the data, or at least not as
presented in the current text.
Please include a full etymology of the two candidatus at the end of the paper, so to protect yourself
against a recent tendency by some people to change
prokaryotic taxonomy (and names), creating a
lot of confusion.
Finally, be extra careful to provide all raw metagenomic data to the readership.
Author Rebuttal to Initial comments
Reviewer #1 (Remarks to the Author):
Overall, this study is a very interesting read and innovative, and the reconstruction of two
complete Heimdallarchaeota genomes is an important contribution to our understanding of the
Asgard archaea. I do believe it deserves to be in
Nature
Micro,
We th
ank
the review for the positive comments on our manuscript and finding it an important
contribution to the field. According to reviewers’ comments, we have restructured and
streamlined the paper to improve focus and clarity to support the main conclusions
of the paper.
however there are some fairly issues that need to be resolved:
The authors state that these complete genomes are “phylogenetically closest to eukaryotes”,
however their phylogenies only include very few of the dozens of genomes available. I
t has
8
been suggested that Heimdall are closest to eukaryotes in various other publications, but this is
not well resolve yet. It is an open question, and a very important one! Their phylogeny supports
this; however, it doesn’t include a comprehensive set n
ow publicly available, there may be other
lineages not included that may be more closely associated with the eukaryote branch. I know
first
-
hand this is not a small undertaking (to resolve the position of eukaryotes) with many
genomes. So, I would back off
on these statements, or run additional phylogenies with more
MAGs.
We acknowledge the complexity in the phylogenetic placement of eukaryotes, and it is an
ongoing effort. After consulting experts who are currently working on this problem, we have now
both sampled a larger range of Asgard archaea lineages to support our co
nclusion and
tempered claims of relatedness with eukaryotes, now better acknowledging that it is still an
ongoing effort.
First, we explained in more detail the existing taxonomy within the Heimdall group archaea that
have influenced the scope of Heimdal
larchaeota, including the drastic differences between the
findings from the recent publications of Liu et al Nature 2021 and Rinke et al Nature
Microbiology 2021. We performed phylogenomic analyses using all publicly available Heimdall
-
related genomes (as
of August 2021) and demonstrated that our circular genomes cluster within
the Heimdall clade. Our updated analysis is now included in Fig. 1b along with companion text
in (
83
-
92
).
To expand our taxonomic sampling, we conducted new analyses with a total of
282 asgard
archaeal genomes available from public databases, including the most recent Liu et al Nature
2021 and Sun et al ISME Comm 2021, which substantially expanded Asgard diversity. We
constructed a phylogenomic tree of all Asgard archaeal genomes usi
ng TACK as outgroup (Fig.
S2). We also constructed new HMMs that performed much better than existing HMMs available
in the CheckM package, which sporadically misses gene markers due to the divergence of
Asgard proteins from the Eury
archaeota
and Crenarchae
ota that were used to construct the old
HMMs. This increased the overall estimates of completeness, as well as redundancy scores for
the Asgard genomes, which we provide as supplementary table 8
and 9
. This is now described
in lines
136
-
139
and in the Meth
ods section. The HMMs are also provided as supplementary
resources on Figshare.
Next
, we applied a less stringent criterion to sample across Asgard lineages with evenness in
mind. The final selected genomes for this analysis are highlighted in supplementa
ry table 9. In
addition to the original tree shown as the main panel in Fig. 1d, we now constructed a new
9
phylogenomic tree based on these selected genomes. This analysis covered majority of Asgard
lineages, although there were a few lineages, Kai, Wukong,
and Jord, lacking high quality
genomes that did not pass our filtering criteria.
For example, Wukongarchaeota from Liu et al
2011 Nature has 3 assemblies of the same species, which all contain nearly 300 contigs.
However, we do not think that it will infl
uence our general claim. Our new tree (Fig. S4) supports
the close relationship between the Heimdall group and eukaryotes. Updated text can be found
in lines
139
-
142
.
Based on these new analyses, we conclude that our phylogeny supports previously reported
placement of Heimdall group as a close relative to eukaryotes, while also acknowledging that
the statistical approach and taxonomic sampling is likely to continue to improve with additional
environmental sequencing of archaea and eukaryotic crown groups i
n the future. We highlight
the value of constructing and evaluating high quality genomes as provided in our study, and
hope that it will play a positive role in future efforts in phylogenomics. Additions to the text can
be found in lines
142
-
146
.
I have
concerns about the identity of some of the CRISPR/Cas
-
containing contigs (shown in
figure 2 and in text ln 203). MAG B51_G16 (GCA_003650225.1) is a Chloroflexi bacteria and
Figure 2f B53_G16 (GCA_003660845.1) is a Pacearchaeota. Both are from NCBI
PRJNA362
212 and associated Guaymas Basin publications. Both of these identities are
supported by Dombrowski et al. 2018 and GTDB. Perhaps these contigs were miss
-
binned in
the original publication, or is there co
-
infection occurring? This is something that should
be
investigated and explained more.
We thank the reviewer for pointing out this discrepancy. The B51_G16 was indeed a typo and
we meant it as B53_G16, which was shown in the figure. We recognize that B53_G16 from
Guaymas Basin is a highly fragmented and i
ncomplete genome, but we found it encodes nearly
identical proteins with our Ca. H. endolithica from Pescadero Basin. We thus performed ANI
analyses and found that in fact it was so close to the latter that they should be classified as the
same species. We
now clarified early on in the text (lines 91
-
94) and re
-
classified this MAG. We
additionally commented on the fact that this genome was recovered from Guaymas basin, a
hydrothermal vent system ~400 km from Pescadero basin in the Gulf of California (line 2
23
-
228).
Sample location for homologs of AagV1
-
7. In Figure 2a (E44_bin34 or GCA_004376455.1)
“homologs of the AagV1
-
7 protein” and in text, pg. 9 and reference 13 is for the Eastern Gulf of
Mexico, Atlantic Ocean (26.28 N 86.81 W)
10
We
have now revised th
e text (line 228
-
231) and within the new figure panel Fig. 3e to indicate
the origin in the Gulf of Mexico.
*****************************************************
Reviewer #2 (Remarks to the Author):
In this manuscript, Wu et al. sequence, assemble and a
nalyse 8 new Asgard archaea genomes,
and perform comparative analyses that bear on archaeal genome evolution and
eukaryogenesis.
The most significant contributions of the manuscript, all of which are in my view of major
importance, are:
(i) the generatio
n, through culture enrichment, of two new complete Heimdallarchaeota
genomes, but the six other high
-
quality new genomes are also very welcome and useful data for
understanding Asgard biology and studying questions around eukaryogenesis;
(ii) comparative genomic analyses of these high
-
quality genomes that confirm previous findings
that Asgards encode various "eukaryote
-
specific" (or eukaryote
-
related) proteins, and may have
donated these to eukaryotes via the Asgard
-
derived host cell for t
he mitochondrial
endosymbiont (Note: though in some respects confirmatory, this kind of confirmation is actually
very important for the field at the moment, given ongoing debates about the quality of the
published Asgard MAGs).
(iii) The identification and
bioinformatic analysis of virus
-
like integrated elements with intriguing
biology in Heimdallarchaeota, and more generally an analysis of the "mobilome" of these
Asgards, which is now possible thanks to the high quality of these assemblies relative to
publ
ished MAGs.
This is an excellent and timely analysis that will make a real difference in ongoing discussions
about eukaryogenesis, but which also provides interesting new data on the genome biology of
Asgards. In general, the methods are appropriate (cave
at: those that I am qualified to judge,
particularly the phylogenetics and comparative genomics) and the interpretations are well
-
founded. Some more detailed/specific comments follow.
We thank the reviewer for the positive comments on the manuscript.
Acco
rding to reviewers’
comments, we have restructured and streamlined the paper to improve focus and clarity to
support the main conclusions of the paper.
1. Lines 289
-
90: The eukaryote root and the placement of the eukaryote nuclear lineage within
the Asgar
d archaea are distinct concepts (with the eukaryote root usually used to refer to ideas
about the deepest split within eukaryotes, not their relation to archaea or bacteria). I suggest
reformulating here to avoid confusion, e.g. "This is in agreement with
recent analyses placing
11
the eukaryotic nuclear lineage/host cell for the mitochondrial endosymbiont within the Asgard
archaea" or "This is in agreement with recent analyses supporting a close specific relationship
between eukaryotes and Heimdallarchaeota",
or similar.
For the same reason, it may be worth
re
-
considering the title of the manuscript to make clear that it is eukaryogenesis/the relationship
of eukaryotes and Archaea, and not the inter
-
relationships among eukaryotic groups, that is
being addresse
d
We thank the reviewer for pointing this out and we agree that the use of ‘eukaryotic root’ is
confusing. The revised manuscript avoids use of this term in the title, abstract, and the main
text, now replaced with more specific descriptions as suggested
throughout.
2. Lines 315
-
322: The parallel drawn between the finding of ESPs and especially bacteria
-
origin
genes acting as a kind of accessory "cloud" of genes and the situation in eukaryotes is
interesting, but needs to be unpacked and set out with gr
eater specificity and detail. In
eukaryotes, the finding that species with fewer genes tend to be enriched for archaeal
-
origin
genes has been explained either by the increased functional importance of archaeal
-
origin
genes (e.g. as informational genes), or
alternatively due to loss of the mitochondrion (and so a
large tranche of bacterial
-
origin genes) in the most reduced eukaryotic lineages such as
Microsporidia. Presumably, the hypothetical mechanistic basis here is the former: that the core
archaeal gene
s are playing essential conserved roles in Asgards, whereas gene acquisitions
from bacteria and ESPs are more niche
-
relevant or generally less conserved. If so, this should
be stated clearly and, if possible, tested against the data presented
in Figure 4.
Do the functions
of the 900 core archaeal genes differ from those of the bacteria
-
origin genes? How does the
situation compare to the "half
-
lives" of horizontally
-
acquired genes more generally, which are
often lost soon after they are acquired (e.g. van Pa
ssel et al.
2008
https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1000059
)
We thank the reviewer for the fantastic suggestion. We have now performed functional analyses
on the three different taxonomic groups, and as shown in our newly reconfigured Fig. 5a, indeed
the three groups have different functional distributions. These re
sults are now detailed in (lines
270
-
283). Regarding whether the comparisons to the ‘half
-
lives’ of horizontally transferred
genes, we find it technically challenging at present, as different from the example provided in
van Passel et al, where many E. col
i strains were compared with each other, we currently only
have 2 closely related Heimdall species here. The strain
-
level diversity in Asgard archaea is
absolutely an interesting aspect to study in the future, and likely will provide further statistical
su
pport for our notion of pan
-
Asgard ERPs. We have discussed the issue of timescale in the
Discussion section (line 346
-
350)
3. In Figure 4(c), I found the schematic of the new hypothesis unclear. The "ancestral Asgard"
12
presumably existed before the first H
eimdall archaea, yet the ancestor is shown as donating
genes along the FECA to LECA stem. Perhaps this would be clearer if the donor were labelled
as "related Asgard archaea" or similar (if this is the intended meaning...).
We have replaced ‘ancestral As
gard’ in the figure by ‘related Asgard archaea’, which is now in
panel Fig. 6c.
4. One of the exciting proposals in the study is that some of the bacterial
-
origin genes in
eukaryotes might have been acquired via the archaeal host, or one of its relatives
(i.e., an
"indirect" acquisition from bacteria). However, the gene trees provided in Extended Figure 10 in
support of these individual cases are, with the exception of (d), perhaps not very compelling.
This is not surprising given the difficulty of inferri
ng robust single gene trees, particularly for
anciently diverged sequences, but these results should perhaps be discussed more cautiously
in the main text. It might be useful to quantify the support for an Asgard origin of these genes in
eukaryotes using a
pproximately
-
unbiased tests, or another appropriate tree selection test. The
likelihood of the ML tree in which the Asgard+eukaryote sequences (or a subset of them) form a
clade to the exclusion of Bacteria could be compared to the ML tree in which the clo
sest
bacterial group forms a clade with the
eukaryotes.
We appreciate the helpful and detailed advice from the reviewer for improving the statistical
support of the proposed ‘indirect acquisition’ scenario.
While the statistical support is weak in
some c
ases, if we look at the detailed placement in the phylogenetic tree (which we now find
can vary depending on the model used), the evolutionary trajectory is quite fascinating, as
Asgard archaea are often the only Archaea taxa that stand between bacteria an
d eukaryotes.
However, given the new analyses and text supporting other findings in our manuscript, we
realized there is no room in this manuscript to sufficiently unpack the logic in the text or highlight
this hypothesis in the abstract. We thus decided
to remove this component in our proposal of
eukaryotic origin in the current revision. We have mentioned it as an interesting direction in the
Discussion section (line 356
-
359). In the future, we hope to expand these analyses to include all
archaea which w
ill offer a more comprehensive study and hopefully a more robust conclusion.
We hope that the reviewer finds this decision a reasonable one.
Minor points:
1. Define "mobilome" in the abstract when first used, or reformulate to avoid this perhaps
field
-
specific term.
13
We have
replaced the term using ‘mobile elements’ throughout, except one mention in the
discussion section, where we fully explain this term.
2. In Extended Figure 8, I think the author of the 2013 study was "Alvarez
-
Ponce".
Thank y
ou.
We
have corrected the author’s name and now placed the SI figure panel in the
main figure Fig. 5b.
*****************************************************
Reviewer #3 (Remarks to the Author):
The paper by Wu and colleagues reports further interesting g
enomic insights into the Asgard
archaea.
They enriched two members of Heimdallarchaeota, currently the clade that is closest to
eukaryotes and named them Ca. Heimdallaerchaeum endolithica and Ca Heimdallarchaeum
aukensis. This allowed them to obtain comp
lete circularized genomes from these two strains.
This is a very important result as isolation of Asgard representatives is one of the highest
priorities in the archaeal field. Presently, as far as I understand, only one enriched and
sequenced member of
Asgard in available (Promethearchaeum).
We thank the
reviewer for the positive comment on the manuscript.
The paper is therefore surely an important one, but my general impression is that it lacks focus,
and does not really know which way to go. Even
the abstract does not really reflect the content,
as a large part of the results is focused on proviruses and mobile elements. I commend the
authors for wanting to make a new scenario for the emergence of eukaryotes, but I fear that this
does not rely stro
ngly on the analyses and is not discussed in detail. In this respect, the very
title of the paper does not reflect the content of the article.
We thank the reviewer for providing critical comments and suggestions to help us improve the
flow of the paper.
We have now restructured the paper to improve clarity and focus as
suggested by the reviewer in their comments below. We also substantially edited the abstract to
reflect the central findings of the manuscript more closely.
The main message to me is th
at the authors obtained two enrichments and they analyzed in
14
detail the mobilome, and this is original enough that the paper could be clearly built around it.
The rest of results are presented as confirmations of previous data (placement with respect to,
e
ukaryotes, presence of ESPs). Moreover, if the authors want to claim that this mobilome is
involved in eukaryogenesis through passage of genes from bacteria to the nascent eukaryotic
cell, this must be supported much more by the data and discussed in depth
, clearly showing that
these mobilomes do vehicle genes from bacteria to eukaryotes (e.g., that some of these genes
are embedded in mobile elements? by one clear phylogeny as main figure?). This could be an
extremely powerful message.
We
have now expand
ed the mobilome section to 3 figures (Fig. 2
-
4). Upon reanalysis using the
most updated viral database IMGVR3 and genome database GTDB, we now report a clearer
phylogenetic signal of viral genes, which relate to bacteriophages infecting Bacteriodota and
Fi
rmicutes (Fig. 4a
-
b) and Fig. S8. We now also provided examples of Heimdallarchaeal mobile
elements garnering genes from bacteria as well as Asgard archaea hosts. Regarding the direct
evidence of ESPs embedded within mobilomes, we currently do not have a c
lear example that
can be highlighted. We believe this is because 1) we are only detecting a small fraction of the
mobilome using our approach and the full spectrum of mobilome features for Asgard archaea
are yet to be revealed, and 2) the genes that were p
assed from bacteria into the
Asgard/eukaryotes at the onset of eukaryogenesis have been there for so long that they may no
longer be part of the present
-
day mobile elements. They may, however, be moving around as
part of their pangenome through other means
such as conjugation and homologous
recombination. The latter is in line with our analyses of ERPs in Fig. 6. We thus decided to not
make the direct link between them in the present text and instead emphasize the importance of
timescale in the Discussion (
line 346
-
350).
Related to ESPs vs mobilome, we now revised the text to state that:
They are likely, or could
have been during their evolutionary history, shuffled as part of their mobilome elements.
’ (lines
326)
We now highlight two independent cases that fall under the umbrella of horizontal transfer:
1) Asgard archaea have bacterial input just like eukaryotes have bacterial input, and they both
scale the same with genome size (Fig. 5b); 2) eukaryote
-
related gen
es in Asgards are only
partially conserved and is genome size
-
dependent (Fig. 5c and Fig. 6a
-
b).
Also, I was disappointed that the authors have two beautiful enrichments in their hands but do
not attempt at more detailed microscopy data. This could be ex
tremely interesting to see for
example, if the same protrusions are observed as in Promethe.
15
We agree that morphological descriptions of these archaea are of high importance and this was
part of our initial efforts. We designed several FISH probes specif
ic for our Heimdallarchaeota,
but have not been able to confidently image the Heimdallarchaeum spp. in our enrichments
unfortunately. We suspect that it may have been due to their physical association with the
rock/sediments, which made them hard to separa
te for imaging. As a side note, we also recently
attempted the use probe Heim1
-
526 described in Salcher et al mSphere 2020, but this probe
appears to target a
Methanolobus
archaea
in our microbial community. We will continue to work
towards imaging these
organisms and hope that further enrichment will allow us to acquire
information about their morphology and ultrastructural organization.
I might be wrong, but I think the authors are not citing nor comparing their results to those of the
recent
Nature
pap
er by Koonin and Meng Li. This is important.
We have
cited their paper in the previous version of the manuscript for their finding of ESPs. In
our revised manuscript, we have fully analyzed all genomes available from their study and
referenced this work.
Finally, I found the figures complex (figure 2 in particular) and not very well explained in the
text.
We
have
now expanded our manuscript from 4 figures to 6 figures and improved their
descriptions in the captions and the text.
Below a few comments
to help improve readability and impact.
This is a personal impression, but I feel that the text is structured somehow erratically.
I think the first thing the readers want to see is a tree where these two genomes are placed, and
this instead comes very
late in Figure 3.
Then I would move on to describe what is new in terms of ESPs in these genomes (basically
move here the paragraph from lines 265
-
303), and then move on to the most original results
(the mobilome and the gene flux), which would nicely le
ad to the proposed hypothesis (although
substantiated by more analysis).
Also, the first paragraph is very hard to follow in the absence of a main figure.
16
We thank the reviewer for helping us streamlining the flow of the manuscript. We have now
accordi
ngly moved the entire section forward and composed a new Figure 1 that is more
reflective of the main message of this section and serves to guide the readers. We have moved
the original text in lines 265
-
303 forward. The new text associated with Figure 1 c
an now be
found in lines 53
-
146.
Line 102, this could be a new paragraph presenting a description of the genome contents. Here,
there is a sentence that could be completed by some discussion: line 116, what is the missing
enzyme for ester
-
linked lipids f
or? what does its absence imply? It is also absent from other
Asgard genomes? The authors mention a mosaic distribution of these enzymes but do not really
discuss it.
We have now expanded the sentence describing the ester
-
linked lipids (lines 126
-
131).
We also
added the mosaic distribution of the genes across Asgard archaea in Fig. 1d.
Line 132: why is this intriguing?
The CRISPR/Cas
systems has never been shown to have any particular global organization in
the genomes, and we find it intriguing that they form this kind of 3
-
fold symmetry. However, as
mentioned above with the restructuring in the current text, we opted to remove this
description
as we felt it was somewhat of a distraction to the flow and allowed us to focus more extensively
on other elements in the manuscript. In the revised version, we also simplified the associated
figure (originally figure 1a), by separating the dis
tribution of CRISPR/Cas and the nontandem
repeats in separate panels in Figure 2).
Line 137: did you try to reassemble by using your method?
The authors unfortunately did not submit the original sequencing reads to the public database.
Line 143: say mo
re clearly that they are from bacteria and mention which bacteria.
A large part of the results presents an extensive analysis of integrated proviruses in the two
circular genomes. This is very interesting and as I said, should be highlighted more in both
the
title and the abstract. Lines 250
-
256, please include a main figure and more detailed results to
support your claims at lines 256
-
258).
17
We have now added mobile elements in the title and substantially streamlined the section
regarding proviruses int
o two sections under CRISPR/Cas
-
guided discovery of
Heimdallarchaeal mobile element (Fig. 3) and Diverse evolutionary origins of Heimdallarchaeal
viruses (Fig. 4). The original lines 250
-
258, now expanded as a full section in line 237
-
267
supported by Fig.
3.
Line 305, the analysis of gene flux scaling is nice and new, and could certainly be further
developed. The possibility that the genome sized of the other MAGs are affected by assembly
problems is not evoked.
We have now expanded the analyses and na
rrative into a separate section (Genome
-
scalable
gene flow across domains, lines 270
-
306) and a separate figure (Fig. 5). We think that providing
functional analyses and a comparison with eukaryotic genomes can provide new insights.
We have now also added Figure S9 to specifically address the pitfall of MAGs with varying
qualities. As shown in the Figure and explained in the text (299
-
301), incomplete and
contaminated genomes completely obscured the constant archaeal fraction.
Line
330: interesting model, which could be merged with the last paragraph as a common
discussion. However, as I said earlier, I do not think it is strongly supported by the data, or at
least not as presented in the current text.
We intended for our hypothesi
zed model be somewhat speculative based on the observation of
extensive gene exchange across domain in the Asgard archaea, as well as the apparent fluidity
of ESPs. The information contained in the new Fig. 5 and Fig. 6 hopefully provide further
support fo
r this idea. Our intension is for this illustration
-
assisted model to help general readers
understand the context and to stimulate future discussions in the field. To better clarify the
hypothetical nature of this, we now added the following in line 338
-
34
0 ‘
We
refer to this
conceptual framework ...’.
Please include a full etymology of the two candidatus
at the end of the paper, so to protect
yourself against a recent tendency by some people to change prokaryotic taxonomy (and
names), creating a lot of confusion.
Thank you for this suggestion.
We have now added an etymology section at the end of the
pap
er.
18
Finally, be extra careful to provide all raw metagenomic data to the readership.
The genomes and metagenomic sequencing reads will be public under the Project No.
PRJNA721962
upon the publication of the paper. It is now indicated in the Data availab
ility
section.
Decision Letter, first revision
:
Dear Fabai, dear Victoria,
Thank you for submitting your revised manuscript "Unique mobile elements and scalable gene flow in
archaea at the prokaryote
-
eukaryote boundary" (NMICROBIOL
-
21051233A). It
has now been seen by
the original referees and their comments are below. As you will see, the reviewers find that the paper
has improved in revision, and therefore we'll be happy in principle to publish it in Nature Microbiology,
pending some minor revisio
ns to satisfy the referees' final requests and to comply with our editorial
and formatting guidelines.
We are now performing detailed checks on your paper and will send you a checklist detailing all our
editorial and formatting requirements once these are
completed. These checks usually take around a
week, but I will be away from the office next week, so will probably only be able to send you all these
documents once I return, on October 25th. Please do not upload the final materials and make any
revisions
until you receive this additional information from us.
Thank you again for your interest in Nature Microbiology, and please do not hesitate to contact me if
you have any questions.
Congratulations once again, and best regards,
{redacted}
************
*****************************************
Reviewer Comments:
Reviewer #1 (Remarks to the Author):
All of my comments have been addressed and I feel it's ready for publication.
*****************************************************
Reviewer #2 (Remarks t
o the Author):
19
Wu et al. responded positively to the first round of revision, and this is an excellent manuscript that
provides valuable new data and presents some conceptually interesting and provocative hypotheses
about the prokaryote
-
to
-
eukaryote trans
ition.
I have two minor points that the authors might wish to consider, both of which could be very quickly
addressed.
1. I think "Andrew Rogers" in the acknowledgements should perhaps read "Andrew Roger", unless this
refers to a less eminent phylogeneti
cist with a confusingly similar name.
2. In the revised manuscript, the section "Genome
-
scaleable gene flow across domains" presents a
very interesting parallel between the chimeric genomes of Asgards and eukaryotes. I think the title of
the section is pe
rhaps difficult to understand, in the sense that this key idea (one of the main
conceptual arguments of the paper) doesn't immediately jump out (since "genome
-
scaleable" is not
very understandable, at least to me). I would suggest the authors might wish to
change it, although
this is of course their decision, not mine.
Something like "Parallels between genome chimerism in Asgard archaea and eukaryotes" or "The
bacterial contribution to gene content scales with genome size in both Asgard archaea and
eukaryo
tes" might get across the idea better.
*****************************************************
Reviewer #3 (Remarks to the Author):
The authors have largely restructured the text, which now reads better and in a clearer way.
I am satisfied with it.
One
important point is to provide not only ethymology but a full taxonomy for the two genomes
(genus, family, order, phylum), so to establish them as the type strains for all Heimdall. Take a look at
other recent papers that did that.
A few remaining typos:
l
ine 47: in the resolution OF archaeal lineages...
line 87: collectively refer to them AS the Heimdall group...
line 92: formerly assigned under the Pacearchaeota, WHICH is...
line 97: and, GIVEN THE ABSENCE OF discernable terminal electron acceptors, MAY d
issipate...
line 116: provide the full taxo too for H repetitus?
line 173: a feature often exploited by bacteriophages
-
> and archaeal viruses too?
lines 241
-
242: Bacteriodota or Bacteroidota?
line 247: While A majority of the viruses...
line 248: Caudivir
ales or Caudovirales?
line 385: I guess it is Andrew Roger not Rogers.
20
Decision Letter,
final checks
:
Dear Fabai, dear Victoria,
Thank you again for your patience as we’ve prepared the guidelines for final submission of your
Nature Microbiology
manuscript, "Unique mobile elements and scalable gene flow in archaea at the
prokaryote
-
eukaryote boundary" (NMICROBIOL
-
21051233A). Please carefully follow the step
-
by
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instructions provided in the attached file, and add a response in each row of the
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