The genome sequence of the Four-dotted Footman, Cybosia mesomella (Linnaeus, 1758)

We present a genome assembly from an individual male Cybosia mesomella (the Four-dotted Footman; Arthropoda; Insecta; Lepidoptera; Erebidae). The genome sequence is 948 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, with the Z sex chromosome assembled. The mitochondrial genome has also been assembled and is 15.4 kilobases in length.


Background
Cybosia mesomella, Four-dotted Footman, is widespread but not particularly common in England and Wales, much more local further North. The only species of the genus Cybosia, it is found across much of the Palaearctic, but not in the far north or in the far south-west. It is not established in Ireland.
The English name references the very distinctive feature of two black dots near the outer and trailing edges of each forewing. In describing Tinea mesomella, (Linnaeus, 1758) emphasised the black centre of the underside of the forewing (Emmet, 1991). Adult moths are most frequently silvery-white with a yellow edge to the forewing, but some are entirely yellow, in Britain most frequently those in the south-east (Waring et al., 2017). The sequenced individual was of the yellow form. Four-dotted Footman flies from June to early August, with larvae over-wintering. They feed on lichen and algae on stems of woody plants in a variety of habitats, from gardens to woodlands and heath. While many of the Footmen have been dramatically increasing in their British range and frequency, as a response to improved air quality, C. mesomella seems to be increasing only slightly (Pescott et al., 2015). As the Four-dotted Footman is the only species in the genus, this genome will be particularly useful for comparative genomics of Erebidae, and Lepidoptera more widely.

Genome sequence report
The genome was sequenced from one male Cybosia mesomella ( Figure 1) collected from a garden in Tonbridge, Kent, UK (latitude 51.19, longitude 0.29). A total of 20-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 43 missing joins or mis-joins and removed 10 haplotypic duplications, reducing the assembly length by 0.45% and the scaffold number by 22.73%.
The final assembly has a total length of 947.6 Mb in 51 sequence scaffolds with a scaffold N50 of 33.2 Mb (Table 1). Most (99.92%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes and the Z sex chromosome (Figure 2- Figure 5; Table 2). Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size. The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 98.8% (single 98.2%, duplicated 0.7%) using the OrthoDB v10 lepidoptera reference set (n = 5,286). While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.

Sample acquisition and nucleic acid extraction
A male Cybosia mesomella specimen (ilCybMeso1) was collected from a garden in Tonbridge, Kent, UK (latitude 51.19, longitude 0.29), using an actinic light. The specimen was collected and identified by Gavin Broad (Natural History Museum) and snap-frozen at -80°C.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilCybMeso1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Thorax tissue 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
A Pacific Biosciences HiFi circular consensus DNA sequencing library was 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 head tissue of ilCybMeso1using the Arima v2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly
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., 2022). 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., 2021), which performed 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   The genome sequence is released openly for reuse. The Cybosia mesomella 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 available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1.

Author information
Members of the Natural History Museum Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.4790042.