The genome sequence of the Orange Footman, Eilema sororcula (Hufnagel, 1766)

We present a genome assembly from an individual male Eilema sororcula (the Orange Footman; Arthropoda; Insecta; Lepidoptera; Erebidae). The genome sequence is 729.4 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.46 kilobases in length. Gene annotation of this assembly on Ensembl identified 21,093 protein coding genes.


Background
The Orange Footman Eilema sororcula had a local distribution in the south and east of the UK until the late 20th century, but like many related lichen-feeding Footman species, it has recently spread northwards and increased spectacularly in both its distribution and abundance (Randle et al., 2019).At the time of writing there has been only a handful of records from Ireland (Moths Ireland, 2023), but it has been recorded across much of Europe and east across Eurasia to Korea and China (GBIF Secretariat, 2023).
The preferred habitats for E. sororcula are mature woodlands where its larvae feed on algae and lichens growing on trees (Henwood et al., 2020).In Britain and Ireland, the adult moth is mostly observed during May and June, with the peak period of observations occurring a few weeks earlier in the year than in the 1970s (Randle et al., 2019).
Here we present a chromosomally complete genome sequence for E. sororcula based on one male specimen from Wytham Woods, Oxfordshire, UK.A genome sequence for E. sororcula will facilitate studies into molecular adaptations to lichen-feeding and contribute to a growing data set of resources for understanding lepidopteran biology.

Genome sequence report
The genome was sequenced from one male Eilema sororcula (Figure 1) collected from| Wytham Woods, Oxfordshire, UK (51.77,.A total of 43-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 93-fold coverage in 10X Genomics read clouds was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 81 missing joins or mis-joins and removed 7 haplotypic duplications, reducing the assembly length by 0.15% and the scaffold number by 60.4%, and increasing the scaffold N50 by 5.25%. The final assembly has a total length of 729.4 Mb in 40 sequence scaffolds with a scaffold N50 of 25.3 Mb (Table 1).Most (99.94%) of the assembly sequence was assigned to 30 chromosomal-level scaffolds, representing 29 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/987424.

Sample acquisition and nucleic acid extraction
A male Eilema sororcula (specimen ID Ox000399, individual ilEilSoro1) was collected in Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2020-05-22, using a light trap.The specimen was collected and identified by Douglas Boyes (University of Oxford) and preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilEilSoro1 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.Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using the 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing.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 and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (10X) instruments.Hi-C data were also generated from tissue of ilEilSoro1 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).One round of polishing was performed by aligning 10X Genomics read data to the 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, 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 Eilema sororcula assembly (GCA_914829495.1) 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.Table 3. Software tools: versions and sources.

Software tool Version
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 • 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.

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Genetics, Lepidoptera I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Reviewer Report 27 February 2024 https://doi.org/10.21956/wellcomeopenres.21742.r72064 © 2024 Galarza J.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Juan Galarza University of Oulu, Oulu, Finland
The study presents the chromosomally complete genome sequence of the Orange Footman moth (Eilema sororcula) based on a male specimen collected from Wytham Woods, Oxfordshire, UK.This species historically had a local distribution in the south and east of the UK until the late 20th century, but recent observations indicate a northward spread and a significant increase in both distribution and abundance.The species has also been recorded across Europe and eastwards to Korea and China.E. sororcula is known for its preference for mature woodlands, where its larvae feed on algae and lichens growing on trees.The chromosomally complete genome sequence was obtained from one male specimen using Pacific Biosciences single-molecule HiFi long reads and 10X Genomics read clouds, with additional scaffolding through chromosome conformation Hi-C data.Manual assembly curation addressed missing joins, mis-joins, and haplotypic duplications.The final assembly comprises 40 sequence scaffolds with a total length of 729.4 Mb, and 99.94% of the sequence assigned to 30 chromosomal-level scaffolds.The study emphasizes that the genome sequence of E. sororcula will contribute to molecular adaptation studies related to lichen-feeding.It adds to the growing dataset of genomic resources for understanding lepidopteran biology.The comprehensive methods employed in the study, including sequencing, assembly, and curation, contribute to the reliability of the genomic data presented.This resource has the potential to further scientific investigations into the ecological and molecular aspects of E. sororcula and enhance our understanding of its expanding distribution and ecological adaptations.
The methods employed in this study are appropriate and technically sound.The researchers have followed established protocols, used recognized tools, and provided comprehensive documentation throughout the experimental procedures.The paper adheres to the standards of genomics research, and the detailed methods section facilitates potential replication by other researchers."Most (99.94%) of the assembly sequence was assigned to 30 chromosomal-level scaffolds" -Although I understand that these sentences are generated by filling placeholders with the real values, I think the word 'most' does not capture that essentially the entire assembly (99.94%) is in chrom-level scaffolds.I would suggest to write "The vast majority of the assembly" or something similar, here and in the abstract.

2.
"While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited." -I don't understand this point.The authors likely produced a primary assembly (and provide alternative contigs in the 'alt assembly'), although this is not precisely mentioned in the methods (HiFiasm has different assembly modes).If so, the primary is likely a mix of both haplotypes, and not 'one haplotype' as stated.This should please be clarified.

3.
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 Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Genome assembly / comparative genomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 2 .
Figure 2. Genome assembly of Eilema sororcula, ilEilSoro1.1: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 729,417,332 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 (51,764,148 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (25,298,203 and 16,955,058 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/ilEilSoro1.1/dataset/CAJZCU01.1/snail.

Figure 5 .
Figure 5. Genome assembly of Eilema sororcula, ilEilSoro1.1:Hi-C contact map of the ilEilSoro1.1 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=IiePitoAQqCad3afW9wBMg.
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 Competing Interests: No competing interests were disclosed.Reviewer Expertise: Bioinformatics, Molecular Ecology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.Reviewer Report 30 June 2023 https://doi.org/10.21956/wellcomeopenres.21742.r61541© 2023 Hiller M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Michael Hiller LOEWE-Centre for Translational Biodiversity Genomics (TBG), Senckenberg Nature Research Society, Frankfurt Am Main, Germany This data note reports a reference-quality genome assembly of Eilema sororcula, together with a gene annotation produced by Ensembl.The generated data and methods used are according to the latest standards.The resulting assembly has a very high quality.I have 3 minor comments: typo: collected from| Wytham Woods 1.