The genome sequence of the small copper, Lycaena phlaeas (Linnaeus, 1760) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual male Lycaena phlaeas (the small copper; Arthropoda; Insecta; Lepidoptera; Lycaenidae). The genome sequence is 420 megabases in span. The whole of the assembly is scaffolded into 24 chromosomal pseudomolecules, with the Z sex chromosome assembled. Gene annotation of this assembly on Ensembl has identified 12,147 protein coding genes.


Background
The small copper or common copper, Lycaena phlaeas, is a colourful butterfly with a vast geographical distribution throughout the Holarctic and North and East Central Africa (Tolman & Lewington, 2008). The species is listed as Least Concern in the IUCN Red List (Europe) (Van Swaay et al., 2009). On the British Isles, L. phlaeas can be found widely distributed in Southern England and Wales, and in smaller numbers in Northern England, Scotland and Ireland. It inhabits a wide range of ecosystems from chalk downlands, heathland, woodland clearings to churchyards and urban localities. Males are territorial and bask in patches of bare ground, on rocks or perching on grass stems. Females lay individual eggs on stems or leaves of host plants such as sorrel (Rumex spp.) or Buckwheats (Polygonaceae spp.) and the larvae overwinter as caterpillars. The number of annual generations are typically two or three, but in southern localities diapause seems to be skipped during warm winters. Contrary to most other Lycaenids, the mymecoxenous L. phlaeas lacks both a dorsal nectar organ and tentacle organs, and its larvae are only weakly attended by ants in behavioural interaction experiments (Fiedler, 1991). The standard haploid karyotype of L. phlaeas consists of 24 autosomes and one sex chromosome (Lorković, 1941), the female is heterogametic (WZ), and the genome size has been estimated as 359.7 Mb (Mackintosh et al., 2019).

Genome sequence report
The genome was sequenced from a single male L. phlaeas collected from Gullane, Scotland, UK (latitude 56.04, longitude -2.82) (Figure 1). A total of 55-fold coverage in Pacific Biosciences single-molecule long reads and 89-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 4 missing/misjoins and removed 1 haplotypic duplication, reducing the assembly size by 0.02%, scaffold number by 4.00% and the scaffold N50 by 3.76%.
The final assembly has a total length of 420 Mb in 25 sequence scaffolds with a scaffold N50 of 18 Mb (Table 1). Of the assembly sequence, 100% was assigned to 24 chromosomal-level scaffolds, representing 23 autosomes (numbered by sequence length), and the Z sex chromosome (Figure 2- Figure 5; Table 2). The assembly has a BUSCO (Simão et al., 2015) v5.1.2 completeness of 98.3% (single 98.0%, duplicated 0.3%, fragmented 0.4%, missing 1.3%) 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.

Gene annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Lycaena phlaeas assembly (GCA_905333005.1; Table 1). The 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)  and OrthoDB (Kriventseva et al., 2008). Prediction tools, CPC2 (Kang et al., 2017) and RNAsamba (Camargo et al., 2020), were used to aid determination of protein coding genes.

Methods
Sample collection, DNA extraction and sequencing A single male L. phlaeas was collected from Gullane, Scotland UK (latitude 56.04, longitude -2.82) using a handnet by Konrad Lohse, University of Edinburgh, and identified by the same individual. The specimen was snap-frozen in liquid nitrogen.
DNA was extracted from the whole organism using the MagAttract HMW DNA Extraction kit, according to the manufacturer's instructions at the Scientific Operations core at the Wellcome Sanger Institute (WSI). Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud sequencing  libraries were then constructed according to the manufacturers' instructions. Sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II and Illumina HiSeq X instruments. Hi-C data were also generated from the whole organism using the Arima v2.0 kit and sequenced on HiSeq X.

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 and corrected using the gEVAL system (Chow et al., 2016) as described previously (Howe et al., 2021). Manual curation was performed using gEVAL, HiGlass (Kerpedjiev et al., 2018) and Pretext. The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2021). 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.

Ethical/compliance issues
The materials that have contributed to this genome note were supplied by a Tree of Life collaborator. The WSI 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; • Legality of collection, transfer and use (national and international).