The genome sequence of the Grey Shoulder-knot, Lithophane ornitopus (Hufnagel, 1766) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual male Lithophane ornitopus (the Grey Shoulder-knot; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 508.6 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.33 kilobases in length. Gene annotation of this assembly on Ensembl identified 18,397 protein coding genes


Background
Lithophane (Lithophane) ornitopus Hufnagel, 1766 (Grey Shoulder-knot) is a member of a widespread Holarctic genus of noctuid moths typically active from autumn (occasionally late summer) to early spring, with a variably prolonged period of rest in sheltered sites during the coldest winter months, depending on the latitude and elevation.Like most of the species within this genus, it is characterised by comparatively long and narrow forewings which have an obviously cryptic pattern, enabling the adult to camouflage against bark.However, the etymology of this species' name refers to another feature of its forewings, namely the strong black trifurcated mark at their base that is reminiscent of bird's foot toes (from the Greek órnis = bird, and poús = foot) (Spuler, 1908).
The moths fly by night and come readily to artificial light and alcohol-sugar lures.The species is univoltine, inhabiting a variety of broad-leaved forested environments, but it is most abundant in oak woodlands, Quercus spp.being its preferred host plants.Other recorded hostplants are Prunus spp., Populus spp., Salix spp.and Ulmus spp.The larvae develop during spring and early summer, usually aestivating before pupation takes place in late summer, in a tough cocoon spun in the soil (Ronkay et al., 2001).
Lithophane (L.) ornitopus is widely distributed through the Palaearctic Region from western Europe and north-west Africa (Morocco) across the Urals and Mediterranean to western Siberia and western Kazakhstan.Records from central Asia should be verified with respect to the closely similar Lithophane (L.) pruinosa (Butler, 1878) substituting L. (L.) ornitopus in east Asia (Kononenko, 2016;Ronkay et al., 2001).
Here we present a chromosomal-level genome assembly for Lithophane ornitopus, based on one male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from a male Lithophane ornitopus (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, -1.34).A total of 58-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 14 missing joins or mis-joins and removed 3 haplotypic duplications, reducing the scaffold number by 5.13%.
The final assembly has a total length of 508.6 Mb in 36 sequence scaffolds with a scaffold N50 of 17.2 Mb (Table 1).
The snail plot 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 (99.98%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 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).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.

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., 2023), 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 Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the

Figure 2 .
Figure 2. Genome assembly of Lithophane ornitopus, ilLitOrni1.1:metrics.The BlobToolKit snail plot 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 508,611,498 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 (23,095,587 bp, shown in red). .Orange and pale-orange arcs show the N50 and N90 scaffold lengths (17,228,122 and 12,539,399 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/CAOKYS01/dataset/CAOKYS01/snail.

Figure 3 .
Figure 3. Genome assembly of Lithophane ornitopus, ilLitOrni1.1:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAOKYS01/dataset/CAOKYS01/blob.

Figure 4 .
Figure 4. Genome assembly of Lithophane ornitopus, ilLitOrni1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAOKYS01/dataset/CAOKYS01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Lithophane ornitopus, ilLitOrni1.1:Hi-C contact map of the ilLitOrni1.1 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=JrYfpw0mQ46ruZibv99pQw.

Table 1 . Genome data for Lithophane ornitopus, ilLitOrni1.1. Project accession data
(Oatley et al., 2023), 2023)arváez-Gómez et al., 2023).HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol(Oatley et al., 2023).The DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 31(Bates et al., 2023).Sheared DNA was purified 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.