Peer review of: Chromosome-level assembly of the horseshoe crab genome provides insights into its (2020)



REVIEWERS' COMMENTS: 

Reviewer #1 (Remarks to the Author): 

This is an interesting manuscript that provides important new insights into the outcomes of whole 
genome duplications in animals. My previous review of the manuscript highlighted a few minor 
concerns that the authors have now addressed. 

Reviewer #2 (Remarks to the Author): 

What a difference! The responses and clarifications by the authors have improved the manuscript 
dramatically. It is now much clearer and argues quite convincingly for three genome doublings in 
the horseshoe crab lineage. The statistics for multimember gene families now included provides 
valuable information. It is actually a rather small fraction of the tick genome that allows 
comparison with the HSC: 5286 genes out of 24,000. Only 56 of these have more than 4 co-
orthologs in HSC. 

However, two matters remain, namely concerning the new Fig. 6 and and Fig. 7, or rather the way 
they are described and interpreted. This should be possible to rectify by adjusting the text. 

Regarding the authors' response to my question about Page 7 and Fig. 5B: 
As the HSC and tick lineages diverged such a long time ago, the situation shown in the figure (now 
Fig. 6a) could have arisen by multiple rearrangements within the tick chromosome (and some in 
the HSC chromosomes), thereby reshuffling gene order. This is quite frequently observed among 
vertebrates lineages. The ancestral genome doublings can nevertheless be deduced in vertebrates 
by comparing many different species, representing deep lineages. This abundance of lineage 
representation is not available for the present comparison. The authors write in conjunction with 
the description of Fig. 6: "This pattern of interdigitated distribution of mangrove HSC orthologues 
on different chromosomes suggests that these syntenic blocks of genes in mangrove HSC are likely 
to be the result of WGD rather than segmental or tandem duplication events." I would suggest the 
authors tune down the argument by replacing "are likely to" to "may". 

Regarding my question about page 8 and Fig. 6 (now Fig. 7), I had written: "Again, why is this 
analysis interpreted to mean up to 8 copies of each block rather than a lot of fissions?" I should 
have written: Why does this indicate 3 WGD events rather than only 1? 
The authors' description of the figure now reads like this: "each mangrove HSC chromosome 
contained paralogons mapping to multiple other chromosomes. This pattern is more consistent 
with WGDs rather than independent chromosomal or tandem gene duplication events and supports 
the hypothesis that the HSC lineage has experienced three rounds of WGD." 
However, I still cannot see how this analysis can say whether there were 1, 2 or 3 WGD events. 
Each region in the HSC genome is shown to one (1) similar region elsewhere in the genome. For 
those (small and few) blocks that have more than one duplicate, how did the analysis choose to 
display only one? And which one? I assume it is the most similar one (= the most recent one) that 
is caught with this analysis. Maybe the blocks from earlier WGD events are so small and few that 
they fall below the threshold of the analysis? As far as I can tell, the analysis displayed in Fig. 7 
shows that the entire HSC genome has been duplicated, but it is impossible to tell from this 
analysis whether it happened more than once. Even the phrase "each mangrove HSC chromosome 
contained paralogons mapping to multiple other chromosomes" I find misleading. I think it would 
be more fair to say: each mangrove HSC chromosomal region displays similarity to one other 
chromosomal region, presumably reflecting the most recent WGD event.