The genome sequence of the Black Lace-weaver spider, Amaurobius ferox (Walckenaer, 1830)

We present a genome assembly from an individual female Amaurobius ferox (the Black Lace-weaver; Arthropoda; Arachnida; Araneae; Amaurobiidae). The genome sequence is 3,564.8 megabases in span. Most of the assembly is scaffolded into 23 chromosomal pseudomolecules, including the X 1, X 2 and X 3 sex chromosomes. The mitochondrial genome has also been assembled and is 14.24 kilobases in length.


Background
Amaurobius ferox (Walckenaer, 1830), commonly known as the Black Lace-weaver, is a species of the Amaurobiidae family.It is one of three Amaurobius species occurring in Britain, which share a characteristic lace-like web (Miller et al., 2010;Řezáč et al., 2017).When viewed dorsally, Amaurobius ferox is very similar to the other two British species of this genus, Amaurobius fenestralis and Amaurobius similis, with markings in females ranging from dusky to ill-defined (Roberts, 1995).All three species have rather wide distributions in Britain, and are found in diverse habitats.There are some differences among them in terms of their micro-habitat preferences.Amaurobius fenestralis prefers to build its webs in woodlands, usually at the soil level (in the leaf litter or under fallen logs and large stones), although it can also be found in crevices of tree bark or in plants with stiff structure and dense foliage (such as hedgerows).Amaurobius similis is perhaps the most familiar species of this genus as it is often found in houses and sheds (even in very urban settings), building its webs in crevices of walls and windows, although it can also share some of the same woodland habitats as A. fenestralis, especially near habitations.Amaurobius ferox prefers to build its webs under stones or rubble and seems to prefer shadier, more humid microhabitats in walls or in tunnels or caves entrances (Bee et al., 2017;Bristowe, 1958;Roberts, 1995).Despite these differences, Amaurobius species are adaptable, and so the location of the web is not a reliable way to distinguish them (Bee et al., 2017;Roberts, 1995).Males of all three species emerge in the same seasons, mostly late summer and autumn, with some males being found in late winter and spring.However, Amaurobius ferox appears to have its male peak emergence season in spring.Phenology is thus also unreliable for distinguishing this species, and analysis of the genitalia is strongly encouraged for accurate separation of the species (Bee et al., 2017).
Amaurobius ferox males have a distinctive pedipalp with two tibial apophysis (one being wide and blunt, the other hook-shaped), females have a distinct epigyne, with a triangular shape where the base is curved inwards and the tip points towards the spinnerets (Roberts, 1995).Maternal care in this sub-social species is highly complex, where the mother produces trophic eggs for her spiderlings (Kim & Roland, 2000), with spiderlings and their mother practising matriphagy (Kim & Horel, 1998).The young sub-social spiders also cooperate in prey capture (Kim et al., 2005), and synchronise their development (Kim, 2001).
Given the intricate reproductive behaviours of A. ferox, decoding its genome offers valuable insights into the genetic underpinnings of these complex patterns.In this study, we present a chromosomally complete genome sequence for Amaurobius ferox, derived from one female specimen collected at Chobham Common, Surrey, UK.

Genome sequence report
The genome was sequenced from one female Amaurobius ferox (Figure 1) collected from Chobham Common, Otter Shaw, Surrey, UK (51.37, -0.59).A total of 31-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 47 missing joins or mis-joins and removed 2 haplotypic duplications, reducing the scaffold number by 6.47%, and increasing the scaffold N50 by 1.35%.
The final assembly has a total length of 3564.8Mb in 288 sequence scaffolds with a scaffold N50 of 153.4 Mb (Table 1).The snailplot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99%) of the assembly sequence was assigned to 23 chromosomal-level scaffolds, representing 20 autosomes and the X 1 , X 2 and X 3 sex chromosomes.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).The X chromosomes were assigned based on synteny to Dolomedes plantarius (GCA_907164885.2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.
The estimated Quality Value (QV) of the final assembly is 61.4 with k-mer completeness of 100.0%, and the assembly has a BUSCO v5.3.2 completeness of 98.2% (single = 93.0%,Metadata for specimens, barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics are given at https://links.tol.sanger.ac.uk/species/336583.

Sample acquisition and nucleic acid extraction
The specimen used for genome sequencing was a female Amaurobius ferox (specimen ID NHMUK014449138, ToLID qqAmaFero1), which was collected from Chobham Common, Otter Shaw, Surrey, UK (latitude 51.37, longitude -0.59) on  spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
RNA was extracted from abdomen tissue of qqAmaFero3 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol (do Amaral et al., 2023).The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Protocols developed by the Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023b).

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from cephalothorax tissue of qqAmaFero2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020).To assess the assembly metrics,  et al., 2021;Simão et al., 2015) were calculated.
Table 3 contains a list of relevant software tool versions and sources.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission    of materials by a Darwin Tree of Life Partner is subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.

INSDC accession
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material There is no doubt that the quality of the genome and the assembly as well as the figures are convincingly of high quality.The authors sufficiently describe sample collection, software used.
Although being part of the DToL, the manuscript would benefit a bit more from highlighting the choice of the species, the initial introduction is already there but it would be nice to known if this is the first chromosome level genome of a spider practicing matriphagy and what effect this complex way of maternal care might have on selection and by that genome evolution.In addition, the results presented in the paper could be more described a bit more thoroughly also in the main text, just naming a few of the assembly statistics etc. and provide some comparisons and conclusion to other genomes.
In the methods I would recommend giving some rationale why three different individuals were used for the sequencing, and if it was checked to cause any problems with the assembly, even if it was an obvious reason like the size of the individual, I would still discuss this drawback.Especially the fact that females were used for the genome sequencing but a male for the RNA sequencing.
Another major issue which was already highlighted by another reviewer is that although each version and software used is indicated, the parameters are not given.As well as the sequencing depth is missing.
Is the rationale for creating the dataset(s) clearly described?We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. Reviewer

Zhi-Sheng Zhang
Southwest University, Chongqing, China This paper presents a genome assembly from an individual female Amaurobius ferox.
The genome sequence is 3564.8megabases in span and is scaffolded into 23 chromosomal pseudomolecules, including the X1, X2 and X3 sex chromosome.The rationale for creating the dataset(s)is clearly described.The protocols are appropriate, and the works are technically sound, there are sufficient details of methods and materials provided to allow replication by others.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
The methods does not include many details to make this fully replicable (on parameters used, etc.).The authors provide links to their standard pipeline, and various protocols, so technically it's fine, just not ideal.
The background provides a concise summary of the habitat and identification of the species.Overall, the content is sufficient for a data note.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?

Figure 2 .
Figure 2. Genome assembly of Amaurobius ferox, qqAmaFero1.1:metrics.The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 3,564,847,194 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (214,309,029 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (153,399,618 and 128,711,411 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the arachnida_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/qqAmaFero1_1/dataset/qqAmaFero1_1/snail.

Figure 3 .
Figure 3. Genome assembly of Amaurobius ferox, qqAmaFero1.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/qqAmaFero1_1/dataset/qqAmaFero1_1/blob.

Figure 4 .
Figure 4. Genome assembly of Amaurobius ferox, qqAmaFero1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/qqAmaFero1_1/dataset/qqAmaFero1_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Amaurobius ferox, qqAmaFero1.1:Hi-C contact map of the qqAmaFero1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=dv9liBZBQZmqK02adfoALw.
Report 15 July 2024 https://doi.org/10.21956/wellcomeopenres.23319.r86923© 2024 Zhang Z.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Proposed standards and metrics for defining genome assembly quality" from Rhie et al. (2021).
(Strickland et al., 2023)ecular weight (HMW) DNA extraction at the WSI includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.In sample preparation, the system with speed setting 30(Todorovic et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation(Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA.The concentration of the sheared and purified DNA was assessed using a Nanodrop

No Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? No Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.