The genome sequence of the Chocolate-tip, Clostera curtula (Linnaeus, 1758) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual female Clostera curtula (the Chocolate-tip; Arthropoda; Insecta; Lepidoptera; Notodontidae). The genome sequence is 512.7 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the W and Z sex chromosomes. The mitochondrial genome has also been assembled and is 15.37 kilobases in length. Gene annotation of this assembly on Ensembl identified 12,251 protein coding genes.


Background
Clostera curtula (Notodontidae: Pygaerinae), also known as the Chocolate-tip, is a moth species first characterised by Carl Linnaeus in the 1758 tenth edition of 'Systema Naturae' (Linnaeus, 1758).The adult moth is medium sized, with a wingspan of 27 to 35 mm.This is a distinctive looking moth, with a buff-hued body, three distinct white crosslines adorning the breadth of its forewings, and a rich, chocolate-brown blotch at the forewing tip, from which it gets its common name.A notable characteristic of this species is the presence of pheromone-producing ring glands, found in the distal half of the eighth/ninth intersegmental membrane (Percy-Cunningham & MacDonald, 1987).
Globally, C. curtula is primarily found throughout Europe, spanning from the western regions to as far east as Russia, and extending northwards up to the southern parts of Scandinavia and southward to the Mediterranean (GBIF Secretariat, 2022).This moth has an uneven distribution in Britain, inhabiting predominantly the southern regions of England and eastern Wales, with isolated populations found in specific areas of Scotland, and it is not recorded from Ireland (Waring et al., 2017).The moth's favoured habitat encompasses woodland areas.Larvae of C. curtula feed primarily on poplar species (Populus), with a preference for aspen (P.tremula), and it also consumes sallow (Salix).In Scotland, aspen is the main food plant, and it is only found in aspen woodlands of significant size and is associated with mature trees (Young, 2001).
The English population has two generations, with adults flying in April and May, and then reappearing in August and September.Conversely, populations in Scotland are singlebrooded, taking flight from June through July (Kimber, 2023).The species overwinters as a pupa in a cocoon spun on the larval foodplant.
The generation of a reference genome for Clostera curtula as part of the Darwin Tree of Life project will serve as a valuable resource for the molecular surveillance of the species.Additionally, it will aid in phylogenetic studies, providing insights into its evolutionary relationships with related species.

Genome sequence report
The genome was sequenced from one female Clostera curtula (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, -1.34).A total of 24-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 77-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 94 missing joins or mis-joins and removed 14 haplotypic duplications, reducing the assembly length by 0.54% and the scaffold number by 67.39%, and increasing the scaffold N50 by 25.04%.
The final assembly has a total length of 512.7 Mb in 30 sequence scaffolds with a scaffold N50 of 18.3 Mb (Table 1).The whole assembly sequence was assigned to 30 chromosomal-level scaffolds, representing 28 autosomes and the W and Z sex chromosomes.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-Figure 5; Table 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.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/987902.

Sample acquisition and nucleic acid extraction
A female Clostera curtula (specimen ID Ox000404, individual ilCloCurt1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2020-05-22 using a light trap.The specimen was collected and identified by Douglas Boyes (University of Oxford) and preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilCloCurt1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Tissue from the abdomen 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.Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using the 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing.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.
RNA was extracted from head and thorax tissue of ilCloCurt1 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.RNA was then eluted in 50 μl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay   et al., 2021;Simão et al., 2015) were calculated.
Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Clostera curtula   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

Figure 2 .
Figure 2. Genome assembly of Clostera curtula, ilCloCurt1.2: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 512,681,271 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 (27,404,194 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (18,278,162 and 11,407,441 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 lepidoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Clostera%20curtula/dataset/ilCloCurt1_2/snail.
kit. Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.SequencingPacific Biosciences HiFi circular consensus and 10X Genomics read cloud 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), Illumina HiSeq 4000 (RNA-Seq) and Illumina NovaSeq 6000 (10X) instruments.Hi-C data were also generated from head and thorax tissue of ilCloCurt1 using the Arima2 kit and sequenced on the HiSeq X Ten instrument.

Figure 5 .
Figure 5. Genome assembly of Clostera curtula, ilCloCurt1.2:Hi-C contact map of the ilCloCurt1.2assembly, 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=TkYvdbn6TRCOaQlzXkVLxg.

Table 3 . Software tools: versions and sources.
Wellcome Sanger Institute -Legal and GovernanceThe 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.
• Legality of collection, transfer and use (national and international) Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.The genome sequence is released openly for reuse.The Clostera curtula 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.Raw data and assembly accession identifiers are reported in Table 1.