The genome sequence of the Mother Shipton moth , Euclidia mi (Clerck, 1759)

We present a genome assembly from an individual male Euclidia mi (the Mother Shipton moth; Arthropoda; Insecta; Lepidoptera; Erebidae). The genome sequence is 2,320 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.6 kilobases in length. Gene annotation of this assembly on Ensembl identified 13,454 protein coding genes.


Background
The Mother Shipton, Euclidia mi (Clerck, 1759) (= Callistege mi), is a day-flying moth in the family Erebidae, distributed widely across Europe and north into Scandinavia (GBIF Secretariat, 2021). In the UK, the moth is most common in the south of England where it is frequently seen in May and June on chalk downland, heathland, woodland rides and flower-rich meadows. The adult moth is most active in sunny weather, but is a weak flyer and is usually seen flitting short distances before settling. The larvae have a series of orange, brown, black and white stripes running the length of the body and feed on clovers, trefoils, and other low-growing plants. Loss or reduction of larval prolegs has occurred in several members of the Erebidae including E. mi; retention of just three pairs of prolegs close to the posterior of the abdomen allows the larva to move by looping in a similar way to Geometridae larvae (Byrne & Moyle, 2019). The species overwinters as a pupa.
The English common name, Mother Shipton, derives from the likeness to a face with exaggerated features traced out in profile by a white line on each forewing. The name seems to have originated in the late 18th century, since Moses Harris called the moth 'the mask' in the 1760s, but revised this to 'the Shipton' in the 1770s (Thornton, 2006). 'Mother Shipton' herself was a reclusive prophet and seller of herbal remedies, born Ursula Sontheil in Knaresborough, Yorkshire, England, in 1488, and the subject of superstition and fear in her lifetime. Although Mother Shipton was already a historical figure when the moth was named, Harris was writing at a time of renewed interest in her life with the release of songs, pantomime and satire referring to her supposed prophecies (Thornton, 2006). The cave where Mother Shipton lived can still be visited today. There is no evidence that the face-like wing markings on the moth are recognised as such by predators; the pattern is more likely a simple case of adaptation for crypsis amongst brown vegetation.
A high-quality genome sequence from E. mi and other day-flying moths will facilitate research into adaptations to a diurnal adult lifestyle, while comparison to data from other Erebidae will enable research into morphological evolution in this taxonomic family.

Genome sequence report
The genome was sequenced from one male Euclidia mi specimen (Figure 1) collected from a grassland area of Wytham Woods (latitude 51.77, longitude -1.33). A total of 34-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 119 missing joins or mis-joins and removed seven haplotypic duplications, reducing the assembly length by 0.92% and the scaffold number by 15.6%.
The final assembly has a total length of 2,320.4 Mb in 303 sequence scaffolds with a scaffold N50 of 87.8 Mb (Table 1). Most (99.4%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes and the Z sex chromosome (Figure 2- Figure 5; Table 2). The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 98.7% (single 96.3%, duplicated 2.5%) using the lepidoptera_odb10 reference set. While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.

Sample acquisition and nucleic acid extraction
Two Euclidia mi specimens (ilEucMixx1 and ilEucMixx2) were collected in Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.77, longitude -1.33) on 30 May 2020 by netting. The specimens were collected and identified by Douglas Boyes (University of Oxford) and snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilEucMixx1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Abdomen 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.
RNA was extracted from head and thorax tissue of ilEucMixx2 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 kit. Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

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 were performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina HiSeq 4000 (RNA-Seq) instruments. Hi-C data were also generated from tissue of ilEucMixx1 using the Arima v2 kit, and sequenced on the HiSeq X Ten instrument.

Genome assembly
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 scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2022). The assembly was checked for contamination and corrected using the gEVAL system (Chow et al., 2016) as  described previously (Howe et al., 2021). Manual curation was performed using gEVAL, HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022). The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2022), which performed annotation using MitoFinder (Allio et al., 2020). The genome was analysed and BUSCO scores generated within the BlobToolKit environment (Challis et al., 2020). Table 3 contains a list of all software tool versions used, where appropriate.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the E. mi assembly (GCA_ 944739405.1). Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).

Alexander Mackintosh
The University of Edinburgh, Edinburgh, Scotland, UK This data note describes a genome assembly of the Mother Shipton moth (Euclidia mi). It begins with a brief description of the ecology and biology of this species as well as the origin of its common name. The results section gives an overview of the data generated and also describes the genome assembly using relevant statistics and figures. Finally, the sampling, sequencing, assembly, and gene annotation steps are described in the Methods section.
The report is concise yet contains enough information for readers to understand how the assembly has been generated.
One interesting detail is that this is one of the largest lepidopteran genome assemblies (2.3 Gb) ever generated. The authors do not comment on this, but I expect that the transposable element content of the genome will be analysed and described in a future publication.
This data note does not require any revisions. My only suggestion would be to include the contig N50 (2.8 Mb) in the main text rather than only in Table 1. Contig N50 is a more representative statistic of assembly contiguity than scaffold N50 (87.8 Mb), which is mostly determined by chromosome size.

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? Yes