The genome sequence of the Lichen Button, Acleris literana (Linnaeus, 1758)

We present a genome assembly from an individual male Acleris literana (the Lichen Button; Arthropoda; Insecta; Lepidoptera; Tortricidae). The genome sequence is 674.9 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 16.4 kilobases in length. Gene annotation of this assembly on Ensembl identified 12,577 protein coding genes.


Background
Acleris literana (Linnaeus, 1758), also known as the Lichen Button or Sprinkled Rough-wing, is a moth in the Tortricidae family.The species' vernacular names reference the cryptic colouration of the forewings, which often exhibits attractive black and blue-green patterning, resembling the surface of a lichen and raised scales giving the wing a 'rough' appearance.Like other members of its genus which overwinter in the adult stage, the species is extremely polymorphic, and forewing colouration can vary in the degree of blue colouring, the quantity and distribution of raised scales, and appearance of brown colouring: in some cases, brown colouration totally replaced the green ground-colour of the forewing (Bradley, 1962).Lichen-like colouring of the forewing is shared with species in other lepidopteran families, such as the noctuids Nyctobrya muralis and Griposia aprilina, or the larva of the geometrid Odontopera bidentata, and is an example of convergent evolution.
The species' high degree of polymorphism may be partially explained by apostatic selection, as copulation and egg-laying occur in spring (Bradley et al., 1973;Elliott et al., 2018), and thus there is strong selective pressure on adults to survive the winter.The dominant foodplant for this species in the British Isles is oak (Quercus), however in captivity the larvae accept a range of other deciduous trees (Bradley et al., 1973;Elliott et al., 2018).Larvae feed between spun leaves from May to June, and pupate between June and July.Adults emerge from August and are on the wing into October, after which they hibernate until they re-emerge in the following spring.The species is found locally across the British Isles, being most abundant in oak woodland (Bradley et al., 1973).Globally, the species is confined to Europe and Asia Minor (Hancock et al., 2015).
A genome sequence for Acleris literana will contribute to our understanding of the genomic basis of polymorphism and the evolution of cryptic coloration in Lepidoptera.The genome of Acleris literana 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 Acleris literana based on one male specimen of the form A. literana ab.squamana (Fabricius, 1775), from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one male Acleris literana (Figure 1) collected from Wytham Woods, Oxfordshire, UK (latitude 51.77, longitude -2.34).A total of 29-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 15 missing joins or mis-joins and removed six haplotypic duplications, reducing the assembly length by 0.38% and the scaffold number by 4.88%.
The final assembly has a total length of 674.9 Mb in 39 sequence scaffolds with a scaffold N50 of 22.0 Mb (Table 1).Most (99.97%) 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/1100899.

Sample acquisition and nucleic acid extraction
A male Acleris literana (ilAclLite1) was reared from a pupa within a leaf spinning collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -2.34) on 22 July 2021 by Liam Crowley (University of Oxford).The adult eclosed on 27 July 2021, when it was identified and snap-frozen on dry ice.
The sample was prepared for DNA extraction in the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilAclLite1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Whole organism tissue was disrupted using a Nippi Powermasher fitted with a BioMasher pestle.DNA was extracted at the WSI Scientific Operations core using the Qiagen MagAttract HMW DNA kit, according to the manufacturer's instructions.

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 the Pacific Biosciences SEQUEL II (HiFi) instrument.Hi-C data were also generated from tissue of ilAclLite1 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 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.
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 was done using Nextflow (Di Tommaso et al., 2017)  Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Acleris literana assembly (GCA_946894065.1).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).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 assembly contains 29 autosomes, the Z chromosome and the mitochondrial genome.It is of high-quality, judged by various metrics, and will be useful to the research community.I have following comments.

INSDC accession Chromosome Size (Mb) GC%
1) The expected haploid chromosome number from previous work (e.g. on karyotypes, if any) could be mentioned.The Z chromosome appears to be several times larger than the autosomes.Would the authors be able to comment on this, perhaps in comparison with other tortricid genomes?
2) The motivation of studying the genomic basis of polymorphism seems rather broad.Perhaps wing pattern polymorphism?3) Adding a reference used for species identification would be useful.4) In "Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-Figure 5; Table 2)," only Figure 5 and Table 2 seem to be relevant to this information.Please consider a more suitable place for other figures.5) Source of the transcriptomic data used to generate genome annotation was not mentioned.

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?
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?Partly Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Population 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 Acleris literana, ilAclLite1.1: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 674,889,359 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 (102,365,968 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (21,986,359 and 15,473,039 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/ilAclLite1.1/dataset/CAMPPL01/snail.

Figure 5 .
Figure 5. Genome assembly of Acleris literana, ilAclLite1.1:Hi-C contact map of the ilAclLite1.1 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=ALGJ1wFpRXSSkRYnS_iRIg.