The genome sequence of the Grey Poplar Bell, Epinotia nisella (Clerck, 1759)

We present a genome assembly from an individual male Epinotia nisella (the Grey Poplar Bell; Arthropoda; Insecta; Lepidoptera; Tortricidae). The genome sequence is 585.0 megabases in span. Most of the assembly is scaffolded into 28 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.44 kilobases in length. Gene annotation of this assembly on Ensembl identified 18,952 protein coding genes.


Background
The Grey Poplar Bell Epinotia nisella (Clerck, 1759) is a moth in the Tortricidae family.The species is highly polymorphic and shows extensive variation in the grey, black, and brown colouration of the forewing (Bradley et al., 1979).The species has a Holarctic distribution and is found across northern Eurasia and northern North America (GBIF Secretariat, 2022).The moth is found across the British Isles in woodland, parks, and damp places where the foodplants, Salix and Populus, occur (Bradley et al., 1979).
The species overwinters as an egg or small larva (Elliott et al., 2018).The larva feeds between April and June in catkins or between spun leaves, and pupation occurs in the larval habitation or leaf-litter, and adults occur from July to September (Bradley et al., 1979;Elliott et al., 2018).The adults fly from dusk and come to light, and rest by day on foliage (Bradley et al., 1979).
A genome of Epinotia nisella will contribute to our understand of the genomic basis of polymorphism, and its evolution within lepidoptera.The genome of Epinotia nisella 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 Epinotia nisella, based on one male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one male Epinotia nisella (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, -1.34).A total of 39-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 9 missing joins or mis-joins and removed 5 haplotypic duplications, reducing the assembly length by 1.49% and the scaffold number by 5.13%.
The final assembly has a total length of 585.0 Mb in 37 sequence scaffolds with a scaffold N50 of 21.3 Mb (Table 1).Most (99.92%) of the assembly sequence was assigned to 28 chromosomal-level scaffolds, representing 27 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/989769.

Sample acquisition and nucleic acid extraction
Two Epinotia nisella specimens (ilEpiNise2 and ilEpiNise1) were collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2020-09-08.The specimens were collected and identified by Douglas Boyes (University of Oxford) and preserved on dry ice.The specimen used for genome sequencing was ilEpiNise2 (specimen ID Ox000953), while ilEpiNise1 (specimen ID Ox000952) was used for Hi-C scaffolding.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilEpiNise2 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 whole organism tissue of ilEpiNise1 using the Arimav2 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 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 Epinotia nisella assembly (GCA_932294315.1) 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

Software tool Version
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.
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 Boyes and colleagues report the genome sequence of a male grey poplar bell Epinotia nisella (Clerck 1759).This species is widely distributed in Britain.Molecular data of this species are scarce prior to this report, and are all COI sequences deposited to the NCBI database.This new genome resource will be very useful for further studies, such as understanding their geographical distribution, population structure, evolutionary relationships with other lepidopterans, and the underlying mechanism contributing to their (morphological) polymorphism.
This genome resource is excellent from the summary statistics, with high BUSCO numbers and sequence continuity (scaffold N50), and majority of sequences contained on the 28 pseudochromosomes (plus mitochondrion).To sum up, this is a valuable contribution.
Is the rationale for creating the dataset(s) clearly described?

Sivasankaran Kuppusamy
Division of Taxonomy and Biodiversity, Entomology Research Institute, Loyola College, Chennai, Tamil Nadu, India The authors have done the whole genome sequence of Epinotia nisella (Clerck, 1759).The authors sequenced over 585.0 million base pairs for the genome assembly using 37 scaffolds.The authors observed 19, 249 gene transcripts and 18,952 protein-coding genes during genome assembly.The standard methods were used for DNA isolation and followed appropriate software for the sequence assembly and annotations.
Overall, there are corrections on this manuscript.The manuscript is accepted scientific standards.

Ann-Marie Waldvogel
University of Cologne, Cologne, Germany This report presents a high-quality genome assembly of the lepidoptera genome of Epinotia nisella , a species that is of scientific interest for its variation in wing pattern coloration.With this, the rational for the generation of high-quality genomic resources is obvious and the genome assembly with furthermore contribute to comparative studies.The assembly has been generated and curated according to standard state-of-the art methodology and fulfills all expected criteria by its summary statistics.
Here are some minor comments: *Lepidopterans are known to be very beautiful, and I highly recommend adding a nicer picture that documents the coloration more clearly.
*Last paragraph of the description of background: "A genome ... will contribute to our understanding of the genomic basis of morphotype polymorphism." 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: ecological genomics, biodiversity 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 Epinotia nisella, ilEpiNise2.1: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 585,001,234 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 (45,621,359 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (21,276,924 and 13,438,000 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/ilEpiNise2.1/dataset/CAKOAI01.1/snail.

Figure 5 .
Figure 5. Genome assembly of Epinotia nisella, ilEpiNise2.1:Hi-C contact map of the ilEpiNise2.1 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=PFvoduDaQYKqY9WV75PHww.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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.

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.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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.