The genome sequence of the spider, Parasteatoda lunata (Clerck, 1757)

We present a genome assembly from an individual female Parasteatoda lunata (spider; Arthropoda; Arachnida; Araneae; Theridiidae). The genome sequence is 1,411.4 megabases in span. Most of the assembly is scaffolded into 12 chromosomal, including the X 1 and X 2 sex chromosomes. The mitochondrial genome has also been assembled and is 14.29 kilobases in length.


Background
The theridiid Parasteatoda lunata (formerly Achaearanea lunata) holds a special place in arachnological history in that it was one of the first spiders to be recorded at a specified locality anywhere in the world. The recorder was Martin Lister (1678) and the locality, Askham Wood, near York (Parker & Harley, 1992:106-107).
Parasteatoda lunata is one of three Parasteatoda species found in the British Isles; the others are P. simulans and P. tepidariorum. The last species is now widely used as a model organism with which to study arachnid and, more generally, chelicerate development (for example, Hilbrant et al., 2012;Oda & Akiyama-Oda, 2020). These, and three Cryptachaea species, comprise a group of British theridiids in which the abdomen is teardrop-shaped and distinctly upturned so that the anterior section appears much higher relative to the carapace, and the spinnerets point downwards (Bee et al., 2020).
Parasteatoda lunata builds a tangled framework of silken threads with sticky globules at the anchor points where the web meets substrate. These globules detain prey until the spider can strike. Typically, the webs are built in shaded, sheltered locations, often in deep crevices in tree trunks or in the cavities of I-shaped metal fence uprights where they are overshadowed by trees. The female always incorporates a leaf or other debris in the web to act as a shelter for her and her multiple grey/ brown egg sacs. The species is common in the Midlands and south-eastern England but absent from Wales and Scotland. It seems to be extending its range northwards with the recent (re)colonisation of central Yorkshire -apparently the first records here since Lister's time.
The genome of Parasteatoda lunata was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland. Here we present a chromosomally complete genome sequence for Parasteatoda lunata based on one female specimen from New Earswick, York. The P. lunata genome will provide an extremely useful comparison to that of the closely related P. tepidariorum (Schwager et al., 2017), as well as the genomes of more distantly related spiders in terms of the evolution of genome content and structure.

Genome sequence report
The genome was sequenced from one female Parasteatoda lunata ( Figure 1) collected from Joseph Rowntree School, New Earswick, UK (53.99, -1.07). 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 64 missing joins or mis-joins and removed 4 haplotypic duplications, reducing the scaffold number 8.38%, and increasing the scaffold N50 by 8.99%.
The final assembly has a total length of 1,411.4 Mb in 338 sequence scaffolds with a scaffold N50 of 114.3 Mb (Table 1).  Most (96.61%) of the assembly sequence was assigned to 12 chromosomal-level scaffolds, representing 10 autosomes and the X 1 and X 2 sex chromosomes. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2- Figure 5; Table 2). The X 1 and X 2 chromosomes were identified based on synteny with Metellina segmentata (GCA_947359465.1) (Henriques & Sivell, 2023). 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 59.4 with k-mer completeness of 100%, and the assembly has a BUSCO v5.3.2 completeness of 98.8% (single = 94.3%, duplicated = 4.5%), using the arachnida_odb10 reference set (n = 2,934).
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/1871986.

Sample acquisition and nucleic acid extraction
Two female Parasteatoda lunata specimens used for genome sequencing (specimen ID NHMUK014537466, individual qqParLuna2) and Hi-C scaffolding (specimen ID NHMUK014537472, individual qqParLuna1) were collected from Joseph Rowntree School, New Earswick, England (latitude 53.99, longitude -1.07) on 2021-06-03. The specimens were collected and identified by Geoff Oxford (University of York) and dry frozen (-80°C).
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The qqParLuna2 sample was weighed and dissected on dry ice. Whole organism tissue was disrupted using a Nippi Powermasher fitted with a BioMasher pestle. High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit. HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30. Sheared DNA was purified by solid-phase reversible immobilisation using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample. The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions. DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences SEQUEL II (HiFi) instrument. Hi-C data were also generated from whole organism tissue of qqParLuna1 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 purge_dups (Guan et al., 2020). The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023). The assembly was checked for contamination and corrected as described previously (Howe et al., 2021). Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022). The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2022), which runs MitoFinder (Allio et al., 2020) or MITOS (Bernt et al., 2013 and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence. 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, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020). This work was done using Nextflow (Di Tommaso et al., 2017) DSL2 pipelines "sanger-tol/readmapping" (Surana et al., 2023a) and "sanger-tol/genomenote" (Surana et al., 2023b). The genome was analysed within the BlobToolKit environment (Challis et al., 2020) and BUSCO scores (Manni 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  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 and • Ethical review of provenance and sourcing of the material • Legality of collection, transfer and use (national and international) The genome sequence is released openly for reuse. The Parasteatoda lunata genome sequencing initiative is part of the Darwin Tree of Life (DToL) project. All raw sequence data and the assembly have been deposited in INSDC databases. The genome will be annotated using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1.

© 2023 Bechsgaard J.
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.

Jesper Bechsgaard
Department of Biology, Aarhus University, Aarhus, Denmark This data note described the sequencing of the reference genome of Parasteatoda lunata. I see a value of using this reference genome in evo-devo, but would appreciate if the authors could be more specific about how they see it can be used in comparison to the related species that already have reference genomes.

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? Yes