The genome sequence of the Tufted Button, Acleris cristana (Denis & Schiffermüller, 1775) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual female Acleris cristana (the Tufted Button; Arthropoda; Insecta; Lepidoptera; Tortricidae). The genome sequence is 562.6 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the W and Z sex chromosomes. The mitochondrial genome has also been assembled and is 16.1 kilobases in length. Gene annotation of this assembly on Ensembl identified 12,598 protein coding genes.


Background
Acleris cristana (the Tufted Button) is a micro-moth in the family Tortricidae. The species has a southerly distribution in Britain and is found throughout mainland Europe. There are also a few records from Japan (GBIF Secretariat, 2023).
A. cristana is probably the most variable species amongst British Lepidoptera with over 130 named forms (Sterling & Parsons, 2018). It is thought that the several of the genes influencing colours of individual pattern elements found on the wings of A. cristana segregate independently, which has resulted in numerous forms with very little gradation between them. In contrast, the closely related species, A. hastiana, which is also very variable, demonstrates many intermediate forms making it difficult to separate out the named forms (Hancock et al., 2015). In A. cristana, although the forewing colour varies significantly, the moth almost always has a distinctive tuft of raised scales in the centre of the forewing, giving rise to the common name of 'Tufted Button'.
The adult moth flies at dusk and in the UK is on the wing from September to mid-April. However, this includes a period of dormancy, from late autumn until early spring, after which the moth awakens to mate. Eggs are laid singly, or in small groups, on twigs of trees in the Rosaceae family, usually blackthorn (Prunus spinosa). The larvae can be found in rolled leaf edges, and later instars are found in spun leaves. The larvae pupate between June to August either in folded leaves, or on the ground in leaf litter (Emmet, 2010). The moth occasionally comes to light but can also be found by beating shrubs in the day.
A genome sequence from A. cristana will be useful for research into colour variation in moths, and more generally for comparative studies across the Lepidoptera. The genome of A. cristana 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 A. cristana based on one female specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one female Acleris cristana ( Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77, -1.34). A total of 37-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 53 missing joins or mis-joins and removed six haplotypic duplications, reducing the assembly length by 0.4% and the scaffold number by 36.62%, and increasing the scaffold N50 by 10.95%.
The final assembly has a total length of 562.6 Mb in 45 sequence scaffolds with a scaffold N50 of 17.8 Mb (Table 1). Most (99.72%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 29 autosomes and the Z and W sex chromosomes. The W and Z chromosomes are similar in size, and are large chromosomes, in keeping with the cytogenetic findings of (Šíchová et al., 2013). The Z chromosome was identified based on alignment with Acleris emargana (GCA_927399475.2) which was assembled from a male sample (Z chromosome only). 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.
The estimated Quality Value (QV) of the final assembly is 65.9 with k-mer completeness of 100%, and the assembly has a   Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/758705.

Sample acquisition and nucleic acid extraction
Two Acleris cristana specimens (specimen number Ox000993 and Ox000832, individuals ilAclCris2 and ilAclCris1) were collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.34) on 2020-11-21 and 2020-08-01 respectively. The specimens were taken from the woodland habitat by Douglas Boyes (University of Oxford) using a light trap. The specimens were identified by the collector and snap-frozen on dry ice.
The sample was prepared for DNA extraction in the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilAclC-ris2 specimen was weighed and dissected on dry ice. 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 Pacific Biosciences SEQUEL

II (HiFi) instrument.
Hi-C data were also generated from whole organism tissue of ilAclCris1 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.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Acleris cristana assembly (GCA_948252455.1). 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).

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.  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 Acleris cristana 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.