The genome sequence of the Clifden nonpareil, Catocala fraxini (Linnaeus, 1758) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual male Catocala fraxini (the Clifden nonpareil; Arthropoda; Insecta; Lepidoptera; Erebidae). The genome sequence is 781 megabases in span. The majority of the assembly (99.99%) is scaffolded into 31 chromosomal pseudomolecules, with the Z sex chromosome assembled. The mitochondrial genome was also assembled, and is 15.6 kilobases in length.


Background
Catocala fraxini (Clifden nonpareil or blue underwing) is a large noctuid moth (75-100 mm wingspan) with zigzag markings on broad silver-grey forewings and striking lilac blue flashes across the hindwings. The moth has been recorded across much of the Palaearctic, from central and northern Europe to Russia, Japan and Korea, primarily in dense woodlands containing stands of its larval food plant aspen, Populus tremula. In the UK, C. fraxini was first recorded in the 1740s at 'Cleifden', Buckinghamshire (now Cliveden, famous for the Profumo scandal); the common name derives from the location plus the French 'non pareil' meaning 'without equal' (Wilkes, 1749).
Through the 1800s and early 1900s, C. fraxini was considered an extreme rarity in the UK with small populations in southern and eastern counties, until it became locally extinct by the 1960s. The moth achieved near mythical status amongst entomologists of the time; P.B.M. Allan was obsessed with the species and wrote that if he ever succeeded in catching the moth he would "go to Fortnum & Mason's and buy rare syrups and syllabubs for it" (Allan, 1947). Sporadic records after 1960 were attributed to occasional dispersing individuals reaching the UK from eastern Europe and Scandinavia (Waring et al., 2003). Since 2010, however, there has been a dramatic increase in UK records and the species is now thought to be breeding in woodlands in the south of England (Randle et al., 2019).
In northern Europe C. fraxini has a single generation per year, overwintering as an egg before larval development from April to July. Late instar larvae have a distinctive fringe of hairs that breaks their outline such that they are effectively camouflaged on woody twigs of the host plant. After pupation, adults emerge around September and have a short flight period; they are strictly nocturnal and can be attracted to sugary substances and to light.
The genome of C. fraxini was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all of the named eukaryotic species in the Atlantic Archipelago of Britain and Ireland. Here we present a chromosomally complete genome sequence for C. fraxini, based on one male specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from one male C. fraxini (Figure 1) collected from Wytham Woods, Oxfordshire (biological vicecounty: Berkshire), UK (latitude 51.772, longitude -1.338). A total of 41-fold coverage in Pacific Biosciences single-molecule long reads and 107-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 12 missing/misjoins, reducing the scaffold number by 23.26%.
The final assembly has a total length of 781 Mb in 33 sequence scaffolds with a scaffold N50 of 27.8 Mb ( Table 1). The majority of the assembly sequence (99.99%) was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes (numbered by sequence length), and the Z sex chromosome (Figure 2- Figure 5; Table 2). The assembly has a BUSCO v5.2.2 (Manni et al., 2021) completeness of 99.0% (single 98.2%, duplicated 0.8%) using the lepidoptera_odb10 reference set. While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.

Sample acquisition and DNA extraction
A single male C. fraxini (ilCatFrax1) was collected from Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.772, longitude -1.338) by Douglas Boyes, UKCEH, using a light trap in woodland. The sample was identified by the same individual, and preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute. The ilCatFrax1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Thorax tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts. Fragment size analysis of 0.01-0.5 ng of DNA was then performed using an Agilent FemtoPulse. High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit. Low molecular weight DNA was    according to the manufacturers' instructions. Sequencing was performed by the Scientific Operations core at the Wellcome Sanger Institute on Pacific Biosciences SEQUEL II (HiFi), Illumina NovaSeq 6000 (10X) and Illumina HiSeq 4000 (RNA-Seq) instruments. Hi-C data were generated from head tissue using the Arima Hi-C+ kit and sequenced on NovaSeq 6000.

Genome assembly
Assembly was carried out with Hifiasm (Cheng et al., 2021); 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 assembly with longranger align, calling variants with freebayes (Garrison & Marth, 2012). The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using SALSA2 (Ghurye et al., 2019). The assembly was checked for contamination as described previously (Howe et al., 2021). Manual curation (Howe et al., 2021) was performed using HiGlass (Kerpedjiev et al., 2018) and Pretext. The mitochondrial genome was assembled using MitoHiFi (Uliano- Silva et al., 2021), which performs annotation using MitoFinder (Allio et al., 2020). The genome was analysed and BUSCO scores generated within the BlobToolKit environment (Challis et al., 2020). Table 3 contains a list of all software tool versions used, where appropriate.

Ethics/compliance issues
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  The genome sequence is released openly for reuse. The C. fraxini 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. The genome will be annotated using the RNA-Seq data and presented through the Ensembl