The genome sequence of the Shaded Pug, Eupithecia subumbrata (Denis & Schiffermüller, 1775) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual male Eupithecia subumbrata (the Shaded Pug; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 496.2 megabases in span. Most of the assembly is scaffolded into 24 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 16.17 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,426 protein coding genes.


Background
The Shaded Pug Eupithecia subumbrata is a small moth, with a forewing 10-12 mm in length, that can have dark forms which are difficult to distinguish from other members of the family Geometridae (Waring et al., 2017).A typical specimen has a narrow forewing with a straight or slightly curved leading edge and a small white spot placed centrally, although this spot may be absent.Darker edges surround a chalky-white ground colour, and there are multiple dark cross-lines and a light brown streak along each of the three main radial veins, however, these markings can be large and obscure the ground colour (Waring et al., 2017).
The core United Kingdom range of Eupithecia subumbrata is limited to south-eastern and southern England excluding Cornwall, but there are small local populations elsewhere including the Isles of Scilly, and parts of northern England, Wales and western Scotland.It is most frequently found on chalk grassland but can be found on roadside verges, salt-marshes, sea-cliffs and in woodland glades (Skinner & Wilson, 2009;Waring et al., 2017).
We present a chromosomally complete genome sequence for Eupithecia subumbrata, based on one male specimen collected in Wytham Woods, Oxfordshire for the Darwin Tree of Life Project.

Genome sequence report
The genome was sequenced from one male Eupithecia subumbrata (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 50-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 4 missing joins or mis-joins and removed 2 haplotypic duplications, reducing the assembly length by 0.99% and increasing the scaffold number by 3.03%. The final assembly has a total length of 496.2 Mb in 33 sequence scaffolds with a scaffold N50 of 24.5 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 (99.89%) of the assembly sequence was assigned to 24 chromosomal-level scaffolds, representing 23 autosomes and the Z sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).Chromosome Z was assigned by alignment to Eupithecia dodoneata (GCA_947044415.1)(Boyes et al., 2023), Eupithecia exiguata (GCA_947086465.1)and Eupithecia insigniata (GCA_947859395.1)(Holland et al., 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.

Sample acquisition and nucleic acid extraction
Eupithecia subumbrata specimens were collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire),

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 ilEupSubu2 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).

Software tool Version
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

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Eupithecia subumbrata assembly (GCA_949316285.1) in Ensembl Rapid Release.

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.
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 Eupithecia subumbrata, ilEupSubu1.1: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 496,202,682 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 (35,696,783 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (24,477,164 and 13,819,472 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/ilEupSubu1_1/dataset/ilEupSubu1_1/snail.

Figure 3 .
Figure 3. Genome assembly of Eupithecia subumbrata, ilEupSubu1.1: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/ilEupSubu1_1/dataset/ilEupSubu1_1/blob.

Figure 4 .
Figure 4. Genome assembly of Eupithecia subumbrata, ilEupSubu1.1: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/ilEupSubu1_1/dataset/ilEupSubu1_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Eupithecia subumbrata, ilEupSubu1.1:Hi-C contact map of the ilEupSubu1.1 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=ABIN1E3KSkm4I0Ecz6Jwdg.

Table 3
contains a list of relevant software tool versions and sources.
• 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.