The genome sequence of the Pinion-spotted Pug, Eupithecia insigniata (Hübner, 1790) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual male Eupithecia insigniata (the Pinion-spotted Pug; Arthropoda; Insecta; Lepidoptera; Geometridae). The genome sequence is 438.1 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 15.52 kilobases in length.


Background
The genus Eupithecia, colloquially known as the 'pug moths', contains over a thousand species of geometrid moth found across Eurasia, Africa and the Americas, and is possibly the largest genus in the family Geometridae (Mironov & Galsworthy, 2014). The genus has a reputation for taxonomic confusion, identification errors and under-recording, since many species are small and drab-coloured with similar markings. Even in a limited geographic region such as Britain and Ireland, with fewer than 50 species, several are difficult to distinguish based on wing pattern alone. One of the striking exceptions is the Pinion-spotted Pug, Eupithecia insigniata (known in older literature by the junior synonym Eupithecia consignata). The moth is distinctive, with silvery wings crossed by scalloped grey-brown lines, three chocolate brown patches on the leading edge of the forewings and a prominent, elongate, discal spot. There is very little variation between individuals.
E. insigniata is rare or very local across southern and central England with recent records coming from a few widely dispersed sites; there are three historic Scottish specimens (NBN Atlas Partnership, 2021). The scarcity is puzzling since the larval food plants are common and widespread. The species also has a sparse and scattered distribution across mainland Europe and the southern parts of Scandinavia; records from further east should be treated with caution due to possible confusion with E. insignioides (GBIF Secretariat, 2022;Mironov & Galsworthy, 2014). A description of the larvae and food plants of E. insigniata was first published by Harpur Crewe (1868), although primary credit should be given to Emma Sarah Hutchinson who supplied Henry Harpur Crewe with the eggs which made his study possible. Hutchinson first caught a live specimen of this moth struggling with a spider in her Herefordshire garden in 1864, and from around that time she bred the species continuously in captivity for over 30 years, studying its life-cycle and supplying professional and amateur entomologists with specimens on which they then published (Holland, 2009;Hutchinson, 1892). In captivity, the moth will lay eggs on apple leaves (Malus sp.) or hawthorn flowers (Crataegus sp.), and the larvae can be reared on the leaves of either plant (Harpur Crewe, 1868;Jackson, 1979;Riley & Prior, 2003). In the wild, larvae are found most frequently on hawthorn, but much remains to be confirmed about ecological requirements of the species (Haggett, 1980). The specimen of E. insigniata from which the current genomic work was undertaken was obtained from a garden habitat where apple and hawthorn grow next to each other; the species is recorded regularly at this site. The adult moth flies for a short period in spring after apple blossom has appeared, usually the end of April or start of May in southern England (NBN Atlas Partnership, 2021); larvae feed until July and the pupae overwinter, with the possibility of overwintering for two years (Sterling, 1985).
The complete genome of Eupithecia insigniata was determined as part of the Darwin Tree of Life project. The assembled genome will contribute to the growing set of resources for studying insect ecology and evolution.

Genome sequence report
The genome was sequenced from one male Eupithecia insigniata ( Figure 1) collected from Wallingford, Oxfordshire, UK (51.6, -1.14). A total of 46-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 37 missing joins or mis-joins and removed 9 haplotypic duplications, reducing the scaffold number by 29.31%, and increasing the scaffold N50 by 0.47%.
The final assembly has a total length of 438.1 Mb in 40 sequence scaffolds with a scaffold N50 of 15.4 Mb ( Table 1). Most (99.95%) 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 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/986977.

Sample acquisition and nucleic acid extraction
A male Eupithecia insigniata (specimen ID Ox002138, ilEupInsi1) was collected using a light trap from a garden in Wallingford, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.6, longitude -1.14) on 2022-05-01. Peter Holland (University of Oxford) collected and identified the specimen. The specimen was snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilEupInsi1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Tissue from the whole organism 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 tissue of ilEupInsi1 that had been set aside, 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.

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: • Ethical review of provenance and sourcing of the material The genome sequence is released openly for reuse. The Eupithecia insigniata 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.