Chromosome-level genome and the identification of sex chromosomes in Uloborus diversus

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1. 

   GigaScience

   Mar 4, 2023

   The orb-web

   This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giad002), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

   Reviewer 1: Jonathan Coddington

   This paper presents the first uloborid spider genome--and it is a chromosome level assembly. Genomes of this family are important because the orb web is supposedly independently and convergently evolved in this group. Although my expertise is not in the technology and informatics of genome sequencing, it appears to be well done.

   Figure 1 A. geniculate -- spelling N. clavipes = T. clavipes Table S1 Number of Componenet Sequences-- typo Text single exon We found a -- typo can be ascribed by -- can be inferred by? an Araneid orb-weaver-- araneid usually not capitalized

   The orb-web

   This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giad002), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

   Reviewer 1: Jonathan Coddington

   This paper presents the first uloborid spider genome--and it is a chromosome level assembly. Genomes of this family are important because the orb web is supposedly independently and convergently evolved in this group. Although my expertise is not in the technology and informatics of genome sequencing, it appears to be well done.

   Figure 1 A. geniculate -- spelling N. clavipes = T. clavipes Table S1 Number of Componenet Sequences-- typo Text single exon We found a -- typo can be ascribed by -- can be inferred by? an Araneid orb-weaver-- araneid usually not capitalized ♂X1X2/♀X1X1X2X2.[48] should be ♂X1X2/♀X1X1X2X2 [48]. You might want to be careful about citing Purcell & Pruitt, see https://purcelllab.ucr.edu/blog6.html and other questions about Pruitt's work.

   Re methods, it would be of interest to know what HMW DNA fragment sizes were (expressed as kb, or mb), although Tape Stations are not very accurate. For people who collect spiders with the intent to yield HMW DNA, such data are important. Data are scarce, so any facts are significant.

   Any homologs of the Pyriform spidroin (PySp) in Acanthoscurria? Piriform silk attachment points are a synapomorphy of araneomorph or "true" spiders. Liphistiomorph and mygalomorph spiders do not (cannot?) make point attachments, and the inability to make point attachments either to substrate or silk-silk point attachments probably constrains/ed the evolution of web architectures in non-araneomorph spiders. Therefore finding homologs to PySp spidroins in non-araneomorph spiders is of great interest to explain araneomorph web architecture diversity.

   Likewise, tubuliform spidroin (TuSp) is probably a synapomorphy of entelegyne spiders, with derived female genitalia--a "flow-though" sperm management system. Eggsacs occur widely in non-entelegyne spiders, so it is a mystery why entelegynes have specialized spigots, glands, and spidroins for the same purpose. Indeed, the particular function of tubuliform silk is not clear. Any thoughts on this? E.g.

   It is good to see attention paid to the mitochondrial genome, as many whole genome studies ignore it. In spiders, early work claimed that tRNA's appeared to be peculiar. Masta and Boore. 2004. The Complete Mitochondrial Genome Sequence of the Spider Habronattus oregonensis Reveals Rearranged and Extremely Truncated tRNAs. Molecular Biology and Evolution, Volume 21, Issue 5, May 2004, Pages 893-902. Any comments on U. diversus tRNAs from that point of view?

   Finally, any comments on evidence for or against the convergent evolution of the orb web? Homology between the pseudoflagelliform and flagelliform spidroins would be pertinent. The intro does raise expectations that some of the macro / larger evolutionary questions will be addressed in the paper, but many, see above, are only cursory or not too much. Perhaps include a sentence in intro acknowledging this, but saying that this paper intends to present the genome and address sex chromosomes, but other topics? For example the sections on some of the spidroins do not extensively discuss comparisons with other spider genomes.

   Reviewer 2: Hui Xiang

   In this study, the authors generated huge genome sequencing data and RNA-seq data and provided a genome assembly with rather complicated merging approach, of a spider with novel phylogenetic position. The genome undoubtedly added novel and important resources for deep understanding of spider evolution. However, there are still severe issues that need to be addressed.

   1. There are huge sequencing data from different samples. However, I don't think that marge of different assemblies is good for a final qualified genome. Given high heterozygosity, that illumina data and ONT data from different individuals is quite difficult to use for assembling a clean genome. As shown in Table 2, assembly by Hify approach is not obviously inferior compared with the merged one, but obviously much better in avoiding redundancy. I strongly suggest that the author adopt the genome assembly of Hify data from one individual, instead of merging two sets of assemblies. Illumina and Nanopore assembly may be helpful in fully deciphering silk proteins.
   2. Proportion of repeats are somewhat affected by the quality of assembly. The high heterozygous genome assembly is complicated merged by diverse batch of data, so the real quality might be not as good as the author described. The quality of repeat is especially hard to evaluate. Hence the statements on genome size (Line 193-200) are not convictive.
   3. About the assembly of RNA-seq data. The authors get huge amounts of data. However, it is not so helpful to obtain novel transcripts if the data is saturated. More importantly, assembly of short reads is even not so useful to obtain long transcripts.
   4. As to whole genome duplication. The authors did not provided solid evidence supporting that WGD occurred in U. diversus genome. They only demonstrated two hox clusters therein. The synteny analysis was quite confusing which is not helpful in confirmation of WGD. They need to provide more solid genome-wide evidence, or otherwise totally downplay the statements.
   5. The identification of the sex chromosome is still vague. The statements are not well organized. The statements and the results are so vague and not convictive. "While 8 of the 10 pseudochromsomes had a median read depth of 40 ± 2, pseudochromosomes 3 and 10 were outliers, with read depths of 36 and 33, respectively." The difference in sequencing depth is rather convictive. As I know the authors sequenced female and male samples. So why they didn't clearly compare the depth of the two sex chromosomes between them and make more evidence? Other:
   6. The information of chromosome-level spider genome are not Incomplete. As I know, there is a black widow genome with chromosome-level. The authors need to added this one.
   7. The authors need to release the sequences of the spidroins the identified and described.

   Reviewer 3: Zhisheng Zhang, Ph.D

   The manuscript GIGA-D-22-00169 presents a chromosome-level genome of the cribellate orb-weaving spider Uloborus diversus. The assembly reinforces evidence of an ancient arachnid genome duplication and identifies complete open reading frames for every class of spidroin gene. And the authors identified the two X chromosomes for U. diversus and identify candidate sex-determining genes.

   The methods of work are well fited to the aims of the study, clearly described, and well written.

   Minor comments:

   1. In the Figure 1B, I noticed that it noted the estimated divergence times of the Araneae, I think there should be add the reference, or detail describe how to do.

   2. There is something wrong with the table format, such as Table1, 2, 5 and Table 6.

   3. Line 70: "chromosome- scale" changes to "chromosome-scale".

   4. Line 147 to lines 148: Line breaks error.

   5. Line 458: "[48]" in the wrong location.

   6. Line 511-512: In the genome of spider Uloborus diversus, which chromosome the genes of "sex lethal (sxl)" and "doublesex (dsx)" located at?

   7. Line 515-516: "The 534 shared sex-linked genes in these three species, 14 are predicted to be DNA/RNA-binding", if these sex-linked genes have difference on RNA level between male and female?

   8. Line 685: "Dovetail Chicago and Dovetail Hi-C Sequencing" should be bold.

   9. Line 764: "We then used the Trinity assembler43 v.2.12.0", the number of 43 may be redundancy.

   10. Some softwares lack the number of RRID, such as line 223 "BRAKER2", line 245 of "NOVOplasty", line 790 of "tRNAscan-SE", line 773 of "RepeatModeler", line 774 of "RepeatMasker", line 797 of "EMBOSS", and so on.

   11. Lines 780 "using the BRAKER 2 pipeline" changes to "using the BRAKER2 pipeline".

   12. Lines 950: "Literature Cited" changes to "Reference".

   13. Lines 952-953: wrong cite. The World Spider Catalog is a web online, the version and the data you accessed from should also added, and the author's name should change to World Spider Catalog.

   Read the original source
2. Version published to 10.1093/gigascience/giad002

   Dec 28, 2022
3. 

   Version published to 10.1101/2022.06.14.495972v2 on bioRxiv

   Jun 28, 2022
4. 

   Version published to 10.1101/2022.06.14.495972v1 on bioRxiv

   Jun 16, 2022