Peer review of: Tracing animal genomic evolution with the chromosomal-level assembly of the freshwater (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Kenny et al. have sequenced and assembled the genome of the freshwater sponge 
Ephydatia muelleri using a PacBio long-read sequencing, Chicago library, and 
Dovetail HiC library. The quality of the assembled genome seems to be at the 
chromosomal level, much better than available sponge genomes. They performed 
many analyses including DNA methylation, gene expressions from four 
developmental stages, and amplicon analysis of associated bacteria. The freshwater 
sponge genome at the chromosomal level will provide a resource on studying early 
diverging groups among animals. However, the authors jumped to conclusions 
without examining the gene expressions on adaptation to freshwater environments. 
In case of the Daphnia genome paper (Colbourne et al. 2010), responsive genes to 
hypoxia environment have been reported with differential expression data. In 
addition, several points need clarification. 
 
Detailed points: 
Line 1: The title is attractive but there is less discussion about animal origins at the 
chromosomal level. Rather, they claim that the genome offers a rich resource for 
future study. 
 
Lines 56-57: The analysis in this manuscript does not offer clarity on the adaptation 
to extreme temperatures and freshwater. Differential gene expression analysis or 
comparative genomic analysis between relative species should be provided. 
 
Lines 98-101: It is unclear how the time tree was constructed in Fig. 1. Although 
they referred to Schuster et al. (2018), it reported the Demospongiae phylogeny 
and did not show the metazoan phylogeny, including the Calcarea, 
Homoscleromorpha, Bilateria, and so on. Supplementary Fig. 2 shows the new 
result of metazoan phylogenetic analysis and likely includes more broad interest 
result. 
 
Line 130: A chromosome-level assembly is not “a complete genome”. 
 
Line 138: It shows that “Flavobacterium spp.” includes multiple species. Is 
Flavobacterium sp. correct? 
 
Lines 140-143: Were there any sequences from the algal symbionts, as shown in 
Supplementary Fig. 41? 
 
Line 153: Supplementary Table 6 shows repeat contents, not gene numbers. It is 

shown in Supplementary Table 10. 
 
Lines 200-202: Were syntenic regions between sponges and ctenophores found? 
This comparison will be also useful to discussions on early animal evolution. 
 
Lines 225-227: Were the genes for DNA methyltransferase any specific characters 
in sponges when they had highly methylated genomes? 
 
Lines 281-286: The common gene losses may happen on transition to freshwater 
but it is unclear why shared gene gains were not surveyed using the same data 
sets. The explanation was found in S7.4 of Supplementary file 1 but more 
duplications in marine lineages do not exclude the possibility of specific gene gains 
in freshwater lineages. 
 
Lines 414-447: The section for host-microbe associations is not likely related to the 
genome assembly at chromosomal level and discussions for the animal evolution. 
 
Fig. 2d: In the chromosome figure, what is shown by black bands? 
 
Fig. 4c: It is difficult to distinguish dots in green and blue. 
 
Fig. 4d: Are almost all of duplicated genes found on a chromosome? 
 
 
 
Reviewer #2: 
Remarks to the Author: 
In the current manuscript Kenny et al present the chromosome level assembly of 
the freshwater sponge E muelleri. This constitutes a useful resource for the 
scientific community interested in sponges and early animal evolution. The datasets 
consist of the genome sequence, synteny blocks, as well as RNA-seq and DNA 
methylome tracks accompanied by a useful and intuitive genome browser. The 
genome size, as well as gene numbers and annotations are in line with what is 
expected from previous work. DNA methylation patterns are similar to what has 
been observed in other invertebrates, and are significantly lower than those of A. 
queenslandica. The authors also provide gene expression datasets of early 
development and characterize the E muelleri microbiome. Even though the study is 
not particularly groundbreaking in any of its aspects, I believe that the presented 
analyses as well as the breadth of accompanying datasets merit publication in 
Nature Communications. My minor suggestions for improvement can be found 
below. 
 

Page 9, line 255: “…suggesting that not all repeats are actively targeted by DNA 
methylation in E. muelleri. 
 
Do the authors suggest that some repeats are indeed actively targeted by 5mC? 
Perhaps it would be interesting to analyse this in more detail. Is there anything 
specific about those actively-targeted repeats? Subfamily, evolutionary age? 
 
Page 11, line 332l: I found the analyses of developmental RNA-seq datasets 
somewhat underwhelming. I believe that these datasets allow for more exploration. 
Firstly it would be useful to describe in more detail the developmental stages 
analysed and why these stages were selected. I think that some description of how 
sponge developmental gene expression correlates with bilaterian and cnidarian 
development would be a valuable addition to the manuscript. It would be especially 
interesting to perform analyses of signalling pathways such as Wnt, transforming 
growth factor β, Notch, Hedgehog and similar pathways (and comment on their 
presence / absence in Ephydatia). I understand that some of this has been 
previously addressed, nevertheless revisiting this in light of a novel sponge 
genome, would be beneficial. 
 
I would also suggest toning down the title (“The genomic basis of animal origins: a 
chromosomal perspective from the sponge Ephydatia muelleri”) given that this is 
not the first sponge genome published and that no major novelties associated with 
“animal origins” are discussed. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
NCOMMS-20-06447 
 
I was specifically asked to assess the microbiome analysis presented in lines 421ff 
and hence restrict my comments to this. 
 
There are two aspects to the microbiome analysis. Firstly, a near complete genome 
of a Flavobacterium species was assembled from the shotgun sequencing data of a 
single sponge individual, and secondly a 16S rRNA gene amplicon study was 
performed on a number of sponge individuals collected in the Northern hemisphere. 
 
The assembly and quality control of the Flavobacterium genome was done 
competently and shows that it likely presents a new species. The only technical 
comment I have here is that the 16S rRNA gene-based phylogeny presented in 
Supp Figure 12 looks to me like a cladogram, rather than an actual phylogram. The 

labels of this figure were also impossible to read. I would have also appreciated a 
more in-depth comparison of this apparent sponge-associated bacterium with free-
living counterparts to reveal potential adaptations to symbiosis, but understand that 
this might be beyond the scope of the current study. Nevertheless, please ensure 
that the near-complete genome is deposited in a public database and provide the 
relevant accession number. 
 
The microbiome analysis was also performed overall competently and I only have 
some minor comments (see below). I am not sure, if the data supports the notion 
that the microbiomes of individual samples were “highly similar” (see Supp info 
page 100, last paragraph and main text line 435) given that the BC dissimilarities 
range from over 40-90% (see Figure S 36A). Please reconsider this conclusion. I 
also think it would be valuable to state in the main text (line 441ff) that the four 
“core” ASV were quite variable in abundance across the samples (perhaps give the 
range of relative abundances). This has implication for the notion given that they 
might be “fundamental for the metabolic function of E. muelleri”. 
 
Minor comments: 
 
Supp. Info page 46: Please refer here and elsewhere to “16S rRNA gene phylogeny” 
instead of “16S phylogeny”. 
 
Supp info page 97: What specifically is meant when sequence “aligned poorly” to 
the Silva reference database. 
 
Supp info page 97: What algorithm was used for taxonomic assignments? 
 
Supp info page 98: Please clarify, if the read counts given and saturation results 
are based on the dataset where the abundant mitochondrial sequence was 
removed. It should. 
 
Supp info page 100: Please make sure that writing is so that relative abundances, 
which are determined here, are not confused with absolute abundances. 
 
Supp info page 100: “Archaea” not “Archaeas”. 
 
Supp info page 100, last sentence: Supp Figure 37A does not show any quantitative 
data on diversity and is therefore not suitable to support the statement made here. 
 
Supp info page 102: Please provide information of what fraction of ASV sequence 
were used for the functional prediction, so that the accuracy can be evaluated. 

Re: Kenny et al, April 28, 2020