The genome sequence of the Birch Marble, Apotomis betuletana (Haworth, 1811)

We present a genome assembly from an individual male Apotomis betuletana (the Birch Marble; Arthropoda; Insecta; Lepidoptera; Tortricidae). The genome sequence is 684 megabases in span. Most of the assembly is scaffolded into 28 chromosomal pseudomolecules with the Z sex chromosome assembled. The mitochondrial genome has also been assembled and is 15.8 kilobases in length. Gene annotation of this assembly on Ensembl identified 21,717 protein coding genes.

A. betuletana was classified as 'common' in a recent assessment of British microlepidoptera (Davis, 2012). Adults fly between June and September in the UK and are readily attracted to light, and traps baited with (Z)-10-tetradecenyl acetate will attract males (Booij & Voerman, 1984). The species was named in 1811 as Tortrix betuletana by Adrian Hardy Haworth in volume III of his 'Lepidoptera Britannica' (Haworth, 1811), with the species name recognising the association with birch (Betula sp.), which is the primary, and possibly only, larval foodplant. Larvae feed in rolled and folded birch leaves during early summer.
The basal two-thirds of the adult wing are dark brown to black, with the distal third white, sometimes with small yellowish or brown spots or patches. The overall appearance, as with several other genera of Tortricidae, is of a bird dropping, and the species is often referred to as a bird dropping mimic. Cott, in his 1940 work on adaptive colouration, considered this phenomenon to be a case of 'special resemblance' (Cott, 1940), but it is actually an example of masquerade. Masquerade is differentiated from crypsis in that the latter makes potential prey items difficult to detect and therefore confuses predators' sensory processes, whereas masquerading individuals resemble inanimate, typically inedible, items, such as sticks, stones, or bird droppings, which confuse predators' cognitive abilities (Skelhorn et al., 2010). Masquerade is most effective when the object being copied is common in the environment (Skelhorn et al., 2011;Skelhorn, 2015), raising the possibility that declines in woodland birds (Burns et al., 2020) may counterintuitively lead to a decline in the abundance of this species.
The genome of A. betuletana will aid research into mechanisms of masquerade in Lepidoptera. Here we present a chromosomally complete genome sequence for A. betuletana, based on one male specimen from Wytham Woods, Oxfordshire.

Genome sequence report
The genome was sequenced from one male Apotomis betuletana ( Figure 1) collected from Wytham Woods, UK (latitude 51.77, longitude -1.33). A total of 34-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 71-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 27 missing or mis-joins and removed seven haplotypic duplications, reducing the scaffold number by 24.05%.
The final assembly has a total length of 684.2 Mb in 60 sequence scaffolds with a scaffold N50 of 24.9 Mb (Table 1). Most (99.63%) 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). The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 98.3% (single 97.5%, duplicated 0.7%) 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 nucleic acid extraction
Two A. betuletana specimens (ilApoBetu1 and ilApoBetu2) were collected in Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.77, longitude -1.33) on 31 August 2020 by netting. The specimens were collected and identified by Douglas Boyes (University of Oxford) and snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI). The ilApoBetu1 sample was weighed and dissected on dry ice. Whole organism 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. Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing. 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 and 10X Genomics read cloud 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) and Illumina NovaSeq 6000 (10X) instruments. Hi-C data were also generated from whole organism tissue of ilApoBetu2 using 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). One round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger ALIGN, calling variants with freebayes (Garrison & Marth, 2012). 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., 2022), which performed annotation using MitoFinder (Allio et al., 2020). The genome was analysed and BUSCO scores were generated within the BlobToolKit environment (Challis et al., 2020). Table 3 contains a list of all software tool versions used, where appropriate.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the A. betuletana assembly (GCA_932273695.1) in Ensembl Rapid Release.   for Z sex chromosome information. Please clarify that the morphology described in the first sentence in the second paragraph under "Background" applies to both males and females or is of the males that were sequenced.