Peer review of: The Seminavis robusta genome provides insights into the evolutionary adaptations of (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The authors report the high quality genome data with numerous analyses of the benthic diatom 
Seminavis robusta. The addition of this benthic diatom to previously reported diatoms genomes 
provides the authors with an interesting framework to study the evolution of the different diatome 
types. The authors report a very thorough analysis presented in a clear and intelligible way. 
The authors notably show that the gene repository of the benthic diatom was enriched by tandem 
duplications from genes having functions related to benthic environments, such as biofilm, interactions 
with bacteria. 
Overall, I do not see any major issue with the analysis; the manuscript was as I said thorough and 
clear. The discussion was balanced and convincing. I also do not see any further analysis that could 
enrich the manuscript as it covers really well the objectives set by the authors as it is. The only thing 
one I might recommend is to move some of the analysis to the supplementary material, particularly I 
was not entirely convinced by the resequencing analysis, so that it might make the manuscript a more 
focused on a particular and interesting topic(s). 

Reviewer #2 (Remarks to the Author): 

This is an interesting manuscript reporting the genome sequence of a benthic diatom, Seminavis 
robusta, and other genomic resources, allowing evolutionary and functional insights on how diatoms 
adapt to and structure benthic habitats. Besides a new genome sequence, this paper also presents 
useful new resources, like a huge transcriptomic effort with many conditions of interest for 
understanding Seminavis ecology, and re-sequencing of 48 strains with potential to unravel selective 
pressures. The analyses on gene family expansion, and particularly the role of tandem gene 
duplications, are particularly convincing in general. There are however many other aspects that should 
be improved, including the quality of the genome assembly, as well as analyses on variable gene 
composition, selection, and evolution of the gene repertoire according to lifestyles versus phylogeny. 

Main points 
The quality of the genome assembly appears relatively low in terms of contiguity compared to current 
standards. The use of a high coverage of long PacBio reads supports the expectation of a more 
continuous sequence. Although this does not impact strongly the main conclusions of the paper, the 
usefulness of the resource may be lessened. Regarding the annotation, it is unclear how the repeat 
detection has affected gene prediction, particularly in light of the conclusions that the S.r. genome has 
large family expansions (see page 3). Can the authors provide an assessment on how this was tested? 
Page 6, the authors noted that “We observed that families with an increasing number of genes tended 
to display higher expression divergence than smaller families (Figure 3A)”. Looking at Fig3A does not 
lead so obviously to the conclusions, it seems that gene families with >4 members have no clear 
trend. Subsequently, it is written that “families showing expression divergence are significantly 
enriched in eukaryote and diatom age classes. Reversely, families showing expression conservation 
are significantly enriched in the species-specific age class”. This would not be coherent with the idea 
that gene duplications have played a role in adaptation in this species. Can the authors explain better 
how these observations can be reconciled? 
From page 10, a long paragraph based on resequencing of multiple strains develops ideas on how the 
Seminavis pan-genome is shaped. This is based mostly on short-read data and assemblies. It is well 
known that these methods lead to incomplete genomes, with the consequence that the variable gene 
complement is inflated. As seen in Figure 4A, the core genome is diminishing to an unrealistic point 
because of this bias. A correct analysis of pan-genome can only be built on high-quality sequences, so 
I suggest that the authors remove most of this and concentrate on the gene novelties appearing in the 
re-sequenced genomes, as those might have been expected in the better quality reference. In addition 

it is written page 11 “many dispensable genes lack functional annotation, the remaining genes are 
enriched in DNA metabolism, zinc fingers, transposable element-derived proteins, and telomeric 
proteins”. These annotations suggest potential retentions of repetitive sequences in the gene set, 
which have to be checked. 
Page 12, an analysis of the purifying and positive selections led to the conclusion that dispensable 
genes are less constrained. This is also hypothesized for genes of unknown functions. In both cases, 
the set is enriched for poorly conserved genes, which may be difficult to annotate, and potentially 
could create cases of poor alignments interpreted as cases of positive selections. The authors should 
show more convincingly that this is not the case, testing for biases potentially introduced in this 
analysis. 
From page 13 onwards, an interesting conservation analysis is made using 88 transcriptomes of very 
diverse diatoms from the MMETSP. The authors found interesting cases of apparent correlation 
between gene presence and some traits, and provide explanations like “benthic signatures” or raphid-
specific traits. This would be reinforced by a clear comparison on how the traits and gene 
amplifications are present in the diatom phylogeny. This would be more insightful than some of the 
present conclusions on rhodopsins and ROS which are expected, and not experimentally shown to be 
linked to the hypotheses. 

Minor point 
Page 5, the diatoms with larger genomes are shown to have more gene families than those with 
smaller genomes. Is this conclusion robust with the way the heterozygosity was treated in the other 
studies? 

Reviewer #3 (Remarks to the Author): 

The manuscript by Osuna-Cruz et al. ostensibly presents the genome of the benthic diatom Seminavis 
robusta. However, the manuscript goes well beyond a genome “announcement” and includes results 
from re-sequencing of 48 S. robusta strains from three clades to identify core genes and strain-
specific genes, analysis of dozens of transcriptomes to enhance gene-model predictions and identify 
gene families induced under specific or multiple conditions, and incorporates comparative analyses 
(including the open Diatom Plaza) to identify genes essential to life in a benthic environment. This is a 
stunning body of work that raises the bar very high for other genome papers and will undoubtedly be 
emulated by others. Therefore, I provide here some detailed suggestions to ensure that others can 
reproduce the results and analyses. I also suggest that the authors discuss their results in a bit more 
detail and provide more comparisons to what is known from other organisms. The discussion of the 
purifying selection is a good example of comparative discussion that should be enhanced in some 
other sections. 

1) Figure 2. Panel A. The authors should not assume that the reader will know what an “upset plot” is 
and should provide more details. For example, what are the black vs gray circles in the rows and what 
do the vertical lines connote? 
2) Figure 3. Panel A. Axis label descriptions in legend are reversed. Panel D. This panel uses a lot of 
symbols/colors. Please make sure all are described. For example, what do the colored boxes indicate? 
The color legend in the center of the figure seems to relate to panels B, C and D, but this is hard to 
understand. 
3) Figure 5. The experimental design was not entirely clear. It was not immediately clear to me that 
the MMETSP data was used only to define the different categories of genes that would be analyzed in 
the Seminavis transcriptomes and that the up/down arrows were derived from these latter analyses. 
4) The authors provide citations for all the software used in their analyses. Please also include all 
settings used to allow others to reproduce the results. 
5) The authors generate additional transcriptomes for this manuscript. They need to provide additional 
details about the growth and harvest conditions. 

6) The authors should provide details about the flow cytometry methods used to estimate genome size 
and possible explanations for the discrepancy in size estimates. 
7) The authors should provide more details on the manual checking used to remove only the 4 
scaffolds out of 33 scaffolds that were designated as potential bacterial contamination. 
8) Figure S3. This figure/legend needs more detail to be interpretable. What are the different lanes for 
each labeled contig? Although expected product sizes are provided in Table S8, it would be helpful to 
include the expected product size in this figure. 
9) Detection of reference-specific genes deserves more discussion. What is the likelihood that these 
genes are truly strain-specific? What sequencing depth of the re-sequenced strains would be required 
to address this question? 
10) The sentence on page 10 beginning with “The six times higher fraction of dispensable genes…” 
requires a reference citation. Also, it is not clear whether the sentence on page 15 beginning with 
“Expression analysis of the GPCR rhodpson-like protein ortholog…” is part of this study or is a 
reference to other work. Please clarify. 
11) Please explain how reference strain chosen and add specificity to DNA isolation techniques. Clarify 
if this strain was used for transcriptome studies and if not used (as it appears), please explain why. 
12) Please note that diatoms have metacaspases, not caspases (page 11). Please correct. 
13) Page 4. The authors state “Newly generated expression data combined with existing data…” Please 
provide references for existing data.