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

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

Gene duplication is thought to generate genetic material to facilitate organismal innovation and 
diversification. It follows that whole genome duplication (WGD) is often thought to have 
contributed to the evolutionary success of animals like vertebrates. However, relatively few WGD 
events have been found so far in animals and therefore it is debated if these events necessarily 
beget diversification or indeed are needed for diversification. Therefore, it is necessary to identify 
WGD events that have occurred during animal evolution, especially independent events from 
vertebrate WGDs, and to assess their potential contribution to diversification. 

Horse-shoe crabs (HSCs) are a very interesting case. There are only four extant species of these 
animals, but it was previously reported that they had been subject to two ancestral WGDs (Kenny 
et al.,). Furthermore, it appeared that these two WGD events in HSCs were independent of a 
single WGD in the ancestor of other chelicerates – the arachnids, spiders and scorpions (Schwager 
et al.,). However previously assemblies of the HSC genomes have been very fragmented and 
assessment of the outcomes of WGD requires analysis of genes synteny. For many species this has 
only recently become possible using combinations of long read technologies and Hi-C and 
therefore this work on the improved assembly of the genome of the mangrove HSC (to basically 
the chromosomal level) is timely and provides important new insights into the biology of these 
animals and the outcomes of WGD more broadly. It is therefore an important study that 
contributes greatly to our understanding of animal evolution. 

The improved assembly provided by the authors allows them to provide a more detailed 
understanding of the outcome of reiterative WGD in HSCs and in fact they find solid evidence for 
the occurrence of not two but potentially three WGDs in these animals. The main evidence for this 
is their finding of five clusters of Hox genes as well as duplicated clusters of Irx genes and 
microRNAs. In addition, comparisons to the tick genome allowed the authors to identify many 
other clusters of genes that have been retained as duplicates since these WGD events. They also 
provide further evidence that these WGD events are independent from WGD in spiders and 
scorpions. GO analysis of duplicated genes in HSC suggests pervasive retention of genes involved 
in immunity and thus new insights into the biology of these animals. Furthermore, population 
genomics analyses allowed the authors to study population history of these animals and pinpoint a 
reduction in the population size consistent with the start of the most recent ice age. 

The paper is well written and makes an important contribution to our understanding of WGDs and 
animal evolution. It offers a key counterpoint to the paradigmic view of WGD leading to 
diversification as is widely held to be the case in vertebrates. This paper will also serve as a 
platform to further explore the consequences of WGD in these animals, other chelicerates and 
other metazoans. 

I have no major concerns with the paper, but as the authors themselves are very careful 
throughout the manuscript to weigh the evidence for two versus three WGDs in HSCs, and since 
this the evidence provided is not completely conclusive, it might better to adjust the title 
accordingly. 

One other minor point is that figure 3 suggests the Hox clusters are known to be intact in spiders 
and scorpions - however one of the clusters of the spider has only been assembled into two 
fragments so far while the Hox clusters of the scorpion are still quite fragmented in the most 
recent assemblies. 

Reviewer #2 (Remarks to the Author): 

General comments 

The manuscript presents a high-quality genome assembly for the mangrove horse-shoe crab, one 
of four extant horse-shoe crab species. The authors argue for three whole genome duplications 
(WGD), meaning that the genome is principally octaploid. However, three main reasons make me 
hesitate to draw such a conclusion: 
1) No gene families are shown with more than five members, except for the mir-71 micro-RNA 
which has seven. This is despite the fact that the three WGD events are deduced to have 
happened 500-135 Mya. By comprison, teleosts that had their three WGD events in the range 550-
300 Mya can have up to eight copies, i.e., a fully 'octaploid' (but diplodized) gene set. Salmonids 
and carps independently experienced a fourth WGD and can have several additional copies beyond 
eight. 
2) Many tandem duplicates, or gene clusters, are described (Fig.8). If this happens with such high 
frequency, perhaps independent duplications followed by translocations may explain some or most 
of the copies interpreted to be the result of WGD events. 
3) The tick chromsomes in Fig. 5 consistently show high similarity to a single HSC chromosome 
each and then dramaticallly lower similarity to other HSC chromosomes. Why would only a single 
HSC chromosome remain reasonably intact after the three proposed WGD events? For comparison, 
consider the analysis of a lamprey genome by Smith et al. 2018 (PMID 29358652) where a similar 
chart in Fig. 4 conspicuously showed that many lamprey super-scaffolds displayed similarity to 2-4 
chicken chromosomes, cautiously interpreted as one WGD, but most likely two WGD events. For 
the vertebrates, these two events took place >500 Mya and the pattern is still quite obvious. Why 
does the HSC genome show only a single chromosome with high similarity to tick in each and 
every case although its proposed WGD events are more recent? 

The manuscript is overall well written and easy to follow, but a few important pieces of information 
are missing and I am not convinced by the overall conclusions, as detailed below. 

Specific points 

Page 3 and Suppl. Fig. 1: The interpretation of the k-mer data is not described in detail and no 
reference is given. Please explain how the K-mer copy number distribution can provide the 
genome size and the heterozygosity (but apparently not the genome doublings). 

Page 5 and Suppl. Fig. 2: The calculated mutation rate must relate to some kind of time estimate 
for the species divergencies. From the fossil record? Please clarify. 

Page 5: The two basal vertebrate WGD events were shown much more clearly by Nakatani et al. 
2007 (PMID 17652425) and Putnam et al. 2008 (18563158) than by Dehal & Boore who showed a 
number of theoretical figures and then only studied the human genome and who did not even 
include human chromosome 7 with the HoxA cluster in their Fig. 7. 

Page 6: The discussion about the Irx gene clusters should mention the possibility that the Sowah-
like gene in IRX-E on Chr4 may have been translocated from either IRX-C or IRX-D. 

Page 7 and Fig. 3B: Likewise, the chromosomes maps may involve a fission resulting in the two 
genes on Chr15 and the two genes in the second block on Chr1. 

Page 7 and Fig. 5A: It is very clear that each tick contig has a 'favorite' HSC chromosome. If 
octaploidization has taken place in HSC, why has always one chromosome been maintained much 
better than the other seven, as I commented in my point 3 above? 

Page 7 and Fig. 5B: It is not easy to see if single genes in Ixodes corresponds to up to six genes 
HSC. Rather, the Ixodes genes seem to have been dispersed by chromosome fissions onto the six 

chromosomes in HSC. 

Page 8 and Fig. 6: Again, why is this analysis interpreted to mean up to 8 copies of each block 
rather than a lot of fissions? 

Page 8 and Fig. 7: How many gene pairs were included in this analysis? My immediate impression 
when seeing the black plot was that there was one extensive duplication phase (WGD) preceded 
by extended duplications over a long period of time. The data described for tandem duplications 
would seem consistent with the possibility of extensive independent duplications rather than 
additional WGD events. 

Page 10, bottom line: the authors write that there is 'strong evidence for three rounds of WGD'. I 
am afraid I am not as convinced as the authors, as described above.