The genome sequence of the Twin-spotted Quaker, Anorthoa munda (Denis & Schiffermüller, 1775) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual male Anorthoa munda (the Twin-spotted Quaker; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 938.6 megabases in span. Most of the assembly is scaffolded into 27 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 22,894 protein coding genes.


Background
The Twin-spotted Quaker (Anorthoa munda) is the only representative of its genus in the UK. This species was formerly placed in the genus Orthosia alongside several similar spring-flying noctuid moth species known in English as "Quakers" or "Drabs", and the validity of the genus Anorthoa has been challenged (Craik, 2018).
Anorthoa munda has a wide distribution in woodland habitats throughout much of Britain and Ireland (Waring et al., 2017). Formerly absent from much of Scotland, it has expanded its distribution there in recent decades, and is also flying significantly earlier in the year than in the 1970s (Randle et al., 2019). Its distribution extends across temperate areas of Europe, Russian and Asia, eastwards to Japan (GBIF Secretariat, 2023).
Anorthoa munda is a variable species, with the ground colour of the wings varying from a pale buff colour to reddish-brown. The two black median dots on each forewing are distinctive but are occasionally reduced or absent. There is a single annual generation, with adults on the wing in March and April when they are attracted to light traps and can be found feeding on the blossom of sallow trees (Salix spp.) (Waring et al., 2017). The caterpillars feed at night on a wide variety of trees and other woody plants, including ash (Fraxinus excelsior), oak (Quercus spp.), poplar (Populus spp.) and willow (Salix spp.) (Henwood et al., 2020). These moths spend the winter as pupae within cocoons formed underground (Henwood et al., 2020).
A genome sequence for Anorthoa munda will contribute to a growing data set of resources for understanding lepidopteran biology. The genome of Anorthoa munda was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland. Here we present a chromosomally complete genome sequence for Anorthoa munda, based on one male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one male Anorthoa munda ( Figure 1) collected from Woods, Oxfordshire, UK (51.77, -1.34). A total of 27-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 75 missing joins or mis-joins and removed 23 haplotypic duplications, reducing the assembly length by 2.15% and the scaffold number by 20.83%, and decreasing the scaffold N50 by 2.74%.
The final assembly has a total length of 938.6 Mb in 38 sequence scaffolds with a scaffold N50 of 32.4 Mb ( Table 1). Most (99.94%) of the assembly sequence was assigned to 27 chromosomal-level scaffolds, representing 26 autosomes and the Z sex chromosome. Chromosomescale 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/988060.

Sample acquisition and nucleic acid extraction
A male Anorthoa munda (specimen ID Ox001097, individual ilAnoMund1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude

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 was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments. Hi-C data were also generated from head tissue of ilAnoMund1 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    et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021)   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 Anorthoa munda 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.